U.S. patent number 6,780,582 [Application Number 09/574,692] was granted by the patent office on 2004-08-24 for arrays of protein-capture agents and methods of use thereof.
This patent grant is currently assigned to Zyomyx, Inc.. Invention is credited to Dana Ault-Riche, Christian Itin, Steffen Nock, Peter Wagner.
United States Patent |
6,780,582 |
Wagner , et al. |
August 24, 2004 |
Arrays of protein-capture agents and methods of use thereof
Abstract
Arrays of protein-capture agents useful for the simultaneous
detection of a plurality of proteins which are the expression
products, or fragments thereof, of a cell or population of cells in
an organism are provided. A variety of antibody arrays, in
particular, are described. Methods of both making and using the
arrays of protein-capture agents are also disclosed. The invention
arrays are particularly useful for various proteomics applications
including assessing patterns of protein expression and modification
in cells.
Inventors: |
Wagner; Peter (Belmont, CA),
Nock; Steffen (Redwood City, CA), Ault-Riche; Dana (Palo
Alto, CA), Itin; Christian (Menlo Park, CA) |
Assignee: |
Zyomyx, Inc. (Hayward,
CA)
|
Family
ID: |
26813220 |
Appl.
No.: |
09/574,692 |
Filed: |
May 17, 2000 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
353555 |
Jul 14, 1999 |
6329209 |
|
|
|
115455 |
Jul 14, 1998 |
6406921 |
|
|
|
Current U.S.
Class: |
506/32; 422/504;
435/287.1; 435/287.2; 435/287.9; 435/288.3; 435/288.4; 435/6.14;
435/6.16; 435/7.1; 436/518; 436/524; 436/525; 436/527; 436/528;
436/532; 436/533; 436/535; 436/536 |
Current CPC
Class: |
B82Y
5/00 (20130101); B82Y 30/00 (20130101); G01N
33/54393 (20130101); G01N 33/551 (20130101); B01J
2219/00605 (20130101); B01J 2219/0061 (20130101); B01J
2219/00612 (20130101); B01J 2219/00617 (20130101); B01J
2219/00619 (20130101); B01J 2219/00621 (20130101); B01J
2219/00626 (20130101); B01J 2219/00635 (20130101); B01J
2219/00637 (20130101); B01J 2219/00644 (20130101); B01J
2219/00659 (20130101); B01J 2219/00702 (20130101); B01J
2219/00725 (20130101); C40B 40/10 (20130101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 33/551 (20060101); G01N
033/543 () |
Field of
Search: |
;435/6,7.1,287.1,287.9,288.3,288.4,287.2
;436/518,524,525,527,528,532,533,535,536 ;422/57 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
2272081 |
|
Nov 1990 |
|
JP |
|
WO 98/39481 |
|
Sep 1998 |
|
WO |
|
WO 99/40434 |
|
Aug 1999 |
|
WO |
|
WO 00/52209 |
|
Sep 2000 |
|
WO |
|
WO 00/53625 |
|
Sep 2000 |
|
WO |
|
WO 00/54046 |
|
Sep 2000 |
|
WO |
|
Other References
Cha et al. Expression of fused protein, human interleukin-2 and
green fluorescent protein, in insect larvae. Annual Meeting of The
American Institute of Chemical Engineers, Los Angeles, CA, Nov.
1997. .
Colliod et al. "Oriented and covalent immobilization of target
molecules of solid supports" Synthesis and application of a
light-activatable and thiol-reactive cross-linking reagent
Bioconjugate Chem. 4:528-536 (1993). .
Dzgoev et al "Microformat imaging ELISA for pesticide
determination" Anal. Chem. 68(19):3364 (1996). .
Ekins "Ligand assays" from electrophoresis to miniaturized
microarrays Clin. Chem. 44(9):2015-2030 (1998). .
Elkins et al. "Multianalyte microspote immunoassay-microanalytical
"compact disk" of the future" Clin. Chem. 37(11):1965-1967 (1991).
.
Jacobson et al. "Fused quartz substrates for microship
electrophoresis" Anal. Chem. 67:2059-2083. .
Jones et al. "Microminizaturized immunoassays using atomic force
microscopy and compositionally patterned antigen arrays" Anal.
Chem. 70(7):1223-1241 (1998). .
Kemeny. Enzyme-linked immunoassays. In Immuno-Chemistry 1 (eds
Johnstone and Turner). pp. 147-175, Nov. 1997. .
Kricka "Miniaturization of analytical systems" Clin. Chem.
44(9):2008-2014 (1998). .
Marks et al. "By-passing immunication--Human antibodies from V-gene
libraries displayed on phage" J. Mol. Biol. 222:581-597 (1991).
.
Martynova e al. "Fabricating of plastic microfluid channels by
imprinting methods" Anal. Chem. 69:4783-47-89 (1997). .
Mauracher et al. Reduction of rubella ELISA background using heat
denatured sample buffer. J. Imminol. Methods. 145:251-254, 1991.
.
Pale-Grosdemange et al. (1991). Formation of self-assembled
monolayers by chemisorption of derivatives of oligo(ethylene
glycol) of structure HS(CH2).sup.11 (OCH2CH2)mOH on gold. J. Am.
Chem. Soc. 113(1):12-20. .
Rowe et al. "Array biosensor for simultaneous identification of
bacterial, viral and protein analytes" Anal. Chem. 71(17):3846-3852
(1999). .
Sigal et al. "A self-assembled monolayer for the binding and study
of histidine-tagged proteins by surface plasmon resonance" Anal.
Chem 68:490-497 (1996). .
Sitzel et al. "Mass-sensing, multianalyte microarray immunoassay
with imaging detection" Clin. Chem. 44(9):2038-2043 (1998). .
Pham, et al. "Human Interleukin-2 Production in Insect
(Trichoplusia ni) Larvae: Effects and Partial Control of
Proteolysis", Biotechnology and Bioengineering vol. 62(2) pp.
175-182; Jan. 20, 1999..
|
Primary Examiner: Chin; Christopher L.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
This application is a divisional of Ser. No. 09/353,555, filed on
Jul. 14. 1999, now U.S. Pat. No. 6,329,209, which is a
continuation-in-part application of application Ser. No.
09/115,455, filed Jul. 14, 1998, now U.S. Pat. No. 6,406,921, which
are incorporated herein by reference in their entirety.
Claims
What is claimed is:
1. A biochip for displaying attached polynucleotides comprising:
(a) a substrate having an oxide or nitride surface; and (b) an
ordered hydrocarbon monolayer formed from first alkyl chains, said
first alkyl chains having proximal and distal ends, said first
alkyl chains interacting with each other to form said ordered
hydrocarbon monolayer, said ordered hydrocarbon monolayer being
attached to said surface through one or more silane residues on
each of said first alkyl chains' proximal ends, said first alkyl
chains' distal ends having a functional group capable of covalently
attaching a polynucleotide to said first alkyl chain, wherein the
biochip further comprises one or more border regions separating two
or more of said immobilization regions to form two or more spaced
apart immobilization regions.
2. A biochip for displaying attached polynucleotide comprising: (a)
a substrate having an oxide or nitride surface; and (b) an ordered
hydrocarbon monolayer formed from first alkyl chains, said first
alkyl chains having proximal and distal ends, said first alkyl
chains interacting with each other to form said ordered hydrocarbon
monolayer, said ordered hydrocarbon monolayer being attached to
said surface through one or more silane residues an each of said
first alkyl chains' proximal ends, said first alkyl chains' distal
ends each having a functional group capable of covalently attaching
a polynucleotide to said first alkyl chain, wherein said ordered
hydrocarbon monolayer is formed from a ratio of said first
mentioned alkyl chains having a first mentioned functional group
for attaching polynucleotides, and second alkyl chains, said second
alkyl chains having proximal and distal ends, said alkyl chains
interacting with each other to form said ordered hydrocarbon
monolayer, said ordered hydrocarbon monolayer being attached to
said surface through one or more second silane residues on each of
said second alkyl chains' proximal ends, and wherein said ordered
hydrocarbon monolayer is made from two or more different kinds of
molecules.
3. The biochip of claim 1 wherein said functional group is one of
hydroxyl, carboxyl, amino, aldehyde, carbonyl, methyl, methylene,
alkene, alkyne, carbonate, aryliodide, or vinyl groups.
4. The biochip of claim 1 wherein said functional group is a
photoactivatable functional group.
5. The biochip of claim 1 wherein the first alkyl chains are the
same length.
6. The biochip of claim 1 wherein the oxide or nitride surface is
selected from the group consisting of silicon oxide titania,
tantalum oxide, silicon nitride, indium tin oxide, magnesium oxide,
alumina, quanz, glass, and silica.
7. The biochip of claim 1 wherein the first alkyl chains are from 8
to 22 carbons in length.
Description
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention relates generally to arrays of
protein-capture agents and methods for the parallel detection and
analysis of up to a large number of proteins in a sample. More
specifically, the present invention relates to proteomics and the
measurement of gene activity at the protein level in cells.
b) Description of Related Art
Although attempts to evaluate gene activity and to decipher
biological processes including those of disease processes and drug
effects have traditionally focused on genomics, proteomics offers a
more direct and promising look at the biological functions of a
cell. Proteomics involves the qualitative and quantitative
measurement of gene activity by detecting and quantitating
expression at the protein level, rather than at the messenger RNA
level. Proteomics also involves the study of non-genome encoded
events including the post-translational modification of proteins,
interactions between proteins, and the location of proteins within
the cell. The structure, function, or level of activity of the
proteins expressed by a cell are also of interest. Essentially,
proteomics involves the study of part or all of the status of the
total protein contained within or secreted by a cell.
The study of gene expression at the protein level is important
because many of the most important cellular processes are regulated
by the protein status of the cell, not by the status of gene
expression. Also, the protein content of a cell is highly relevant
to drug discovery efforts since most drugs are designed to be
active against protein targets.
Measuring the mRNA abundances of a cell provides only an indirect
and incomplete assessment of the protein content of a cell. The
level of active protein that is produced in a cell is often
determined by factors other than the amount of mRNA produced. For
instance, both protein maturation and protein degradation are
actively controlled in the cell and a protein's activity status can
be regulated by post-translational modifications. Studies comparing
mRNA transcript abundances to protein abundances have found only a
limited correlation (coefficient of about 0.43-0.48) between the
two (Anderson and Anderson, Electrophoresis, 19:1853-1861, 1998).
Furthermore, the extreme lability of RNA in samples due to chemical
and enzymatic degradation makes the evaluation of genetic
expression at the protein level more practical than at the mRNA
level.
Current technologies for the analysis of proteomes are based on a
variety of protein separation techniques followed by identification
of the separated proteins. The most popular method is based on
2D-gel electrophoresis followed by "in-gel" proteolytic digestion
and mass spectroscopy. Alternatively, Edman methods may be used for
the sequencing. This 2D-gel technique requires large sample sizes,
is time consuming, and is currently limited in its ability to
reproducibly resolve a significant fraction of the proteins
expressed by a human cell. Techniques involving some large-format
2D-gels can produce gels which separate a larger number of proteins
than traditional 2D-gel techniques, but reproducibility is still
poor and over 95% of the spots cannot be sequenced due to
limitations with respect to sensitivity of the available sequencing
techniques. The electrophoretic techniques are also plagued by a
bias towards proteins of high abundance.
Standard assays for the presence of an analyte in a solution, such
as those commonly used for diagnostics, for instance, involve the
use of an antibody which has been raised against the targeted
antigen. Multianalyte assays known in the art involve the use of
multiple antibodies and are directed towards assaying for multiple
analytes. However, these multianalyte assays have not been directed
towards assaying the total or partial protein content of a cell or
cell population. Furthermore, sample sizes-required to adapt such
standard antibody assay approaches to the analysis of even a
fraction of the estimated 100,000 or more different proteins of a
human cell and their various modified states are prohibitively
large. Automation and/or miniaturization of antibody assays are
required if large numbers of proteins are to be assayed
simultaneously. Materials, surface coatings, and detection methods
used for macroscopic immunoassays and affinity purification are not
readily transferable to the formation or fabrication of
miniaturized protein arrays.
Miniaturized DNA chip technologies have been developed (for
example, see U.S. Pat. Nos. 5,412,087, 5,445,934, and 5,744,305)
and are currently being exploited for the screening of gene
expression at the mRNA level. These chips can be used to art
determine which genes are expressed by different types of cells and
in response to different conditions. However, DNA biochip
technology is not transferable to protein-binding assays such as
antibody assays because the chemistries and materials used for DNA
biochips are not readily transferable to use with proteins. Nucleic
acids such as DNA withstand temperatures up to 100.degree. C., can
be dried and re-hydrated without loss of activity, and can be bound
physically or chemically directly to organic adhesion layers
supported by materials such as glass while maintaining their
activity. In contrast, proteins such as antibodies are preferably
kept hydrated and at ambient temperatures are sensitive to the
physical and chemical properties of the support materials.
Therefore, maintaining protein activity at the liquid-solid
interface requires entirely different immobilization strategies
than those used for nucleic acids. The proper orientation of the
antibody or other protein at the interface is desirable to ensure
accessibility of their active sites with interacting molecules.
With miniaturization of the chip and decreased feature sizes, the
ratio of accessible to non-accessible and the ratio of active to
inactive antibodies or proteins become increasingly relevant and
important.
Thus, there is a need for the ability to assay in parallel a
multitude of proteins expressed by a cell or a population of :cells
in an organism, including up to the total set of proteins expressed
by the cell or cells.
SUMMARY OF THE INVENTION
The present invention is directed to arrays of protein-capture
agents and methods of use thereof that satisfy the need to assay in
parallel a multitude of proteins expressed by a cell or population
of cells in an organism, including up to the total protein content
of a cell.
In one embodiment, the present invention provides an array of
protein-capture agents comprising: a substrate; at least one
organic thinfilm covering some or all of the surface of the
substrate; and a plurality of patches arranged in discrete, known
regions on the portions of the substrate surface covered by organic
thinfilm, wherein (i) each patch comprises protein-capture agents
immobilized on the organic thinfilm, where the protein-capture
agents of a given patch are capable of binding a particular
expression product, or a fragment thereof, of a cell or population
of cells in an organism; and (ii) the array comprises a plurality
of different protein-capture agents, each of which is capable of
binding a different expression product, or fragment thereof of the
cell or population of cells in the organism.
In a second embodiment, the invention provides an array of bound
proteins which comprises both the array of protein-capture agents
of the invention and a plurality of different proteins which are
expression products, or fragments thereof, of a cell or population
of cells in an organism, where each of the different proteins is
bound to a protein-capture agent on a separate patch of the
array.
Methods of using the arrays of protein-capture agents of the
invention are also provided. In one embodiment of the invention, a
method of assaying in parallel for a plurality of different
proteins in a sample which are expression products, or fragments
thereof, of a cell or a population of cells in an organism, is
provided which comprises first delivering the sample to the array
of protein-capture agents of the invention under conditions
suitable for protein binding, wherein each of the proteins being
assayed is a binding partner of the protein-capture agent of at
least one patch on the array. The final step comprises detecting,
either directly or indirectly, for the presence or amount of
protein bound to each patch of the array. This method optionally
further comprises the step of further characterizing the proteins
bound to at least one patch of the array.
In another embodiment of the invention, a method for determining
the protein expression pattern of a cell or a population of cells
in an organism is provided which comprises first delivering a
sample containing the expression products, or fragments thereof, of
the cell or population of cells to the array of protein-capture
agents of the invention under conditions suitable for protein
binding. The final step comprises detecting, either directly or
indirectly, for the presence or amount of protein bound to each
patch of the array. In an alternative embodiment, a similar method
for comparing the protein expression patterns of two cells or
populations of cells is also provided.
In still another embodiment of the invention, an alternative method
of assaying in parallel for a plurality of different proteins in a
sample which are expression products, or fragments thereof, of a
cell or a population of cells in an organism is provided. The
method of this embodiment comprises first contacting the sample
with an array of spatially distinct patches of different
protein-capture agents under conditions suitable for protein
binding, wherein each of the proteins being assayed is a binding
partner of the protein-capture agent of at least one patch on the
array. The last step of the method involves detecting, either
directly or indirectly, for the presence or amount of protein bound
to each patch of the array.
In a still further embodiment, a method of producing an array of
protein-capture agents is provided which comprises the following
steps: selecting protein-capture agents from a library of
protein-capture agents, wherein the protein-capture agents are
selected by their binding affinity to the proteins from a cellular
extract or body fluid; producing a plurality of purified samples of
the selected protein-capture agents; and immobilizing the
protein-capture agent of each different purified sample onto an
organic thinfilm on a separate patch on the substrate surface to
form a plurality of patches of protein-capture agents on discrete,
known regions of the surface of a substrate.
In an alternative embodiment, the invention provides a method for
producing an array of protein-capture agents which comprises a
first step of selecting protein-capture agents from a library of
protein-capture agents, wherein the protein-capture agents are
selected by their binding affinity to proteins which are the
expression products, or fragments thereof, of a cDNA expression
library. The second step of the method comprises producing a
plurality of purified samples of the protein-capture agents
selected in the first step. The third step comprises immobilizing
the protein-capture agent of each different purified sample onto an
organic thinfilm on a separate patch on the substrate surface to
form a plurality of patches of protein-capture agents on discrete,
known regions of the surface of a substrate.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the top view of an array of patches reactive towards
protein-capture agents.
FIG. 2 shows the cross section of an individual patch of the array
of FIG. 1.
FIG. 3 shows the cross section of a row of monolayer-covered
patches of the array of FIG. 1.
FIG. 4 shows a thiolreactive monolayer on a substrate.
FIG. 5 shows an aminoreactive monolayer on a coated substrate.
FIG. 6 shows the immobilization of a protein-capture agent on a
monolayer-coated substrate via an affinity tag.
FIG. 7 shows the immobilization of a protein-capture agent on a
monolayer-coated substrate via an affinity tag and an adaptor.
FIG. 8 shows a schematic of a fluorescence detection unit which may
be used to monitor binding of proteins by the protein-capture
agents of the array.
FIG. 9 shows a schematic of an ellipsometric detection unit which
may be used to monitor binding of proteins by the protein-capture
agents of the array.
DETAILED DESCRIPTION OF THE INVENTION
A variety of arrays of protein-capture agents and methods useful
for multianalyte analyses and analyses of protein expression and
modification in cells are provided by the present invention.
(a) Definitions
The term "protein-capture agent" means a molecule or a
multi-molecular complex which can bind a protein to itself.
Protein-capture agents preferably bind their binding partners in a
substantially specific manner. Protein-capture agents with a
dissociation constant (K.sub.D) of less than about 10.sup.-6 are
preferred The protein-capture agent will most typically be a
biomolecule such as a protein or a polynucleotide. The biomolecule
may optionally be a naturally occurring, recombinant, or synthetic
biomolecule. Antibodies or antibody fragments are highly suitable
as protein-capture agents. Antigens may also serve as
protein-capture agents, since they are capable of binding
antibodies. A receptor which binds a protein ligand is another
example of a possible protein-capture agent. For instance,
protein-capture agents are understood not to be limited to agents
which only interact with their binding partners through noncovalent
interactions. Protein-capture agents may also optionally become
covalently attached to proteins which they bind. For instance, the
protein-capture agent may be photocrosslinked to its binding
partner following binding.
The term "binding partner" means a protein which is bound by a
particular protein-capture agent, preferably in a substantially
specific manner. In some cases, the protein-capture agent may be a
cellular or extracellular protein and the binding partner may be
the entity, normally bound in vivo. In other embodiments, however,
the binding partner may be the protein or peptide on which the
protein-capture agent was selected (through in vitro or in vivo
selection) or raised (as in the case of antibodies). A binding Ad
partner may be shared by more than one protein-capture agent. For
instance, a binding partner which is bound by a variety of
polyclonal antibodies may bear a number of different epitopes. One
protein-capture agent may also bind to a multitude of binding t
partners, for instance, if the binding partners share the same
epitope.
A "protein" means a polymer of amino acid residues linked together
by peptide bonds. The term, as used herein, refers to proteins,
polypeptides, and peptides of any size, structure, or function.
Typically, however, a protein will be at least six amino acids
long. Preferably, if the protein is a short peptide, it will be at
least about 10 amino acid residues long. A protein may be naturally
occurring, recombinant, or synthetic, or any combination of these.
A protein may also be just a fragment of a naturally occurring
protein or peptide. A protein may be a single molecule or may be a
multi-molecular complex. The term protein may also apply to amino
acid polymers in which one or more amino acid residues is an
artificial chemical analogue of a corresponding naturally occurring
amino acid. An amino acid polymer in which one or more amino acid
residues is an "unnatural" amino acid, not corresponding to any
naturally occurring amino acid, is also encompassed by the use of
the term "protein" herein.
A "fragment of a protein" means a protein which is a portion of
another protein For instance, fragments of a proteins may be a
polypeptides obtained by digesting full-length protein isolated
from cultured cells. A fragment of a protein will typically
comprise at least six amino acids. More typically, the fragment
will comprise at least ten amino acids. Preferably, the fragment
comprises at least about 16 amino acids.
An "expression product" is a biomolecule, such as a protein, which
is produced when a gene in an organism is expressed. An expression
product may optionally comprise post-translational
modifications.
The term "antibody" means an immunoglobulin, whether natural or
partially or wholly synthetically produced. All derivatives thereof
which maintain specific binding ability are also included in the
term; The term also covers any protein having a binding domain
which is homologous or largely homologous to an immunoglobulin
binding domain. These proteins may be derived from natural sources,
or partly or wholly synthetically produced. An antibody may be
monoclonal or polyclonal. The-antibody may be a member of any
immunoglobulin class, including any of the human classes: IgG, IgM,
IgA, IgD, and IgE. Derivatives of the IgG class, however, are
preferred in the present invention.
The term "antibody fragment" refers to any derivative of an
antibody which is less than full-length. Preferably, the antibody
fragment retains at least a significant portion of the full-length
antibody's specific binding ability. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab').sub.2, scFv, Fv,
dsFv diabody, and Fd fragments. The antibody fragment may be
produced by any means. For instance, the antibody fragment may be
enzymatically or chemically produced by fragmentation of an intact
antibody or it may be recombinantly produced from a gene encoding
the partial antibody sequence. Alternatively, the antibody fragment
may be wholly or partially synthetically produced. The antibody
fragment may optionally be a single chain antibody fragment.
Alternatively, the fragment may comprise multiple chains which are
linked together, for instance, by disulfide linkages. The fragment
may also optionally be a multimolecular complex. A functional
antibody fragment will typically comprise at least about 50 amino
acids and more typically will comprise at least about 200 amino
acids.
Single-chain Fvs (scFvs) are recombinant antibody fragments
consisting of only the variable light chain (V.sub.L) and variable
heavy chain (V.sub.H) covalently connected to one another by a
polypeptide linker. Either V.sub.L or V.sub.H may be the NH.sub.2
-terminal domain. The polypeptide linker may be of variable length
and composition so long as the two variable domains are bridged
without serious steric interference. Typically, the linkers are
comprised primarily of stretches of glycine and serine residues
with some glutamic acid or lysine residues interspersed for
solubility.
"Diabodies" are dimeric scFvs. The components of diabodies
typically have shorter peptide linkers than most scFvs and they
show a preference for associating as dimers.
An "Fv" fragment consists of one V.sub.H and one V.sub.L domain
held together by noncovalent interactions. The term "dsFv" is used
herein to refer to an Fv with an engineered intermolecular
disulfide bond to stabilize the V.sub.H -V.sub.L pair.
A "F(ab').sub.2 " fragment is an antibody fragment essentially
equivalent to that obtained from immunoglobulins (typically IgG) by
digestion with an enzyme pepsin at pH 4.0-4.5. The fragment may be
recombinantly produced.
A "Fab'" fragment is an antibody fragment essentially equivalent to
that obtained by reduction of the disulfide bridge or bridges
joining the two heavy chain pieces in the F(ab')2 fragment. The
Fab' fragment may be recombinantly produced.
A "Fab" fragment is an antibody fragment essentially equivalent to
that obtained by digestion of immunoglobulins (typically IgG) with
the enzyme papain. The Fab fragment may be recombinantly produced.
The heavy chain segment of the Fab fragment is the Fd piece.
A "population of cells in an organism" means a collection of more
than one cell in a single organism or more than one cell originally
derived from a single organism. The cells in the collection are
preferably all of the same type. They may all be from the same
tissue in an organism, for instance. Most preferably, gene
expression in all of the cells in the population is identical or
nearly identical. "Conditions suitable for protein binding" means
those conditions (in terms of salt concentration, pH, detergent,
protein concentration, temperature, etc.) which allow for binding
to occur between an immobilized protein-capture agent and its
binding partner in solution. Preferably, the conditions are not so
lenient that a significant amount of nonspecific protein binding
occurs.
A "body fluid" may be any liquid substance extracted, excreted, or
secreted from an organism or tissue of an organism. The body fluid
need not necessarily contain cells. Body fluids of relevance to the
present invention include, but are not limited to, whole blood,
serum, urine, plasma, cerebral spinal fluid, tears, sinovial fluid,
and amniotic fluid.
An "array" is an arrangement of entities in a pattern on a
substrate. Although the pattern is typically a two-dimensional
pattern, the pattern may also be a three-dimensional pattern.
A "patch of protein-capture agents" means a discrete region of
immobilized protein-capture agents on the surface of a substrate.
The patches may be of any geometric shape or may be irregularly
shaped. For instance, the patch may be, but need not necessarily
be, square in shape.
"Proteomics" means the study of or the characterization of either
the proteome or some fraction of the proteome. The "proteome" is
the total collection of the intracellular proteins of a cell or
population of cells and the proteins secreted by the cell or
population of cells. This characterization most typically includes
measurements of the presence, and usually quantity, of the proteins
which have been expressed by a cell. The function, structural
characteristics (such as post translational modification), and
location within the cell of the proteins may also be studied.
"Functional proteomics" refers to the study of the functional
characteristics, activity level, and structural characteristics of
the protein expression products of a cell or population of
cells.
The term "substrate" refers to the bulk underlying, and core
material of the arrays of the invention.
The terms "micromachining" and "microfabrication" both refer to any
number of techniques which are useful in the generation of
microstructures (structures with feature sizes of sub-millimeter
scale). Such technologies include, but are not limited to, laser
ablation, electrodeposition, physical and chemical vapor
deposition, photolithography, and wet chemical and dry etching.
Related technologies such as injection molding and LIGA (X-ray
lithography, electrodeposition, and molding) are also included.
Most of these techniques were originally developed for use in
semiconductors, microelectronics, and Micro-ElectroMechanical
Systems (MEMS) but are applicable to the present invention as
well.
The term "coating" means a layer that is either naturally or
synthetically formed on or applied to the surface of the substrate.
For instance, exposure of a substrate, such as silicon, to air
results in oxidation of the exposed surface. In the case of a
substrate made of silicon, a silicon oxide coating is formed on the
surface upon exposure to air. In other instances, the coating is
not derived from the substrate and may be placed upon the surface
via mechanical, physical, electrical, or chemical means. An example
of this type of coating would be a metal coating that is applied to
a silicon or polymer substrate or a silicon nitride coating that is
applied to a silicon substrate. Although a coating may be of any
thickness, typically the coating has a thickness smaller than that
of the substrate.
An "interlayer" is an additional coating or layer that is
positioned between the first coating and the substrate. Multiple
interlayers may optionally be used together. The primary purpose of
a typical interlayer is to aid adhesion between the first coating
and the substrate. One such example is the use of a titanium or
chromium interlayer to help adhere a gold coating to a silicon or
glass surface. However, other possible functions of an interlayer
are also anticipated. For instance, some interlayers may perform a
role in the detection system of the array (such as a semiconductor
or metal layer between a nonconductive substrate and a
nonconductive coating).
An "organic thinfilm" is a thin layer of organic molecules which
has been applied to a substrate or to a coating on a substrate if
present. Typically, an organic thinfilm is less than about 20 nm
thick. Optionally, an organic thinfilm may be less than about 10 nm
thick. An organic thinfilm may be disordered or ordered. For
instance, an organic thinfilm can be amorphous (such as a
chemisorbed or spin-coated polymer) or highly organized (such as a
Langmuir-Blodgett film or self-assembled monolayer). An organic
thinfilm may be heterogeneous or homogeneous. Organic thinfilm
which are monolayers are preferred. A lipid bilayer or monolayer is
a preferred organic thinfilm. Optionally, the organic thinfilm may
comprise a combination of more than one form of organic thinfilm.
For instance, an organic thinfilm may comprise a lipid bilayer on
top of a self-assembled monolayer. A hydrogel may also compose an
organic thinfilm. The organic thinfilm will typically have
functionalities exposed on its surface which serve to enhance the
surface conditions of a substrate or the coating on a substrate in
any of a number of ways. For instance, exposed functionalities of
the organic thinfilm are typically useful in the binding or
covalent immobilization of the protein-capture agents to the
patches of the array. Alternatively, the organic thinfilm may bear
functional groups (such as polyethylene glycol (PEG)) which reduce
the non-specific binding of molecules to the surface. Other exposed
functionalities serve to tehter the tinfoil to the surface of the
substrate or the coating. Particular functionalities of the organic
thinfilm may also be designed to enable certain detection
techniques to be used with the surface. Alternatively, the organic
thinfilm may serve the purpose of preventing inactivation of a
protein-capture agent or the protein to be bound by a
protein-capture agent from occurring upon contact with the surface
of a substrate or a coating on the surface of a substrate.
A "monolayer" is a single-molecule thick organic thinfilm. A
monolayer may be disordered or ordered. A monolayer may optionally
be a polymeric compound, such as a polynonionic polymer, a
polyionic polymer, or a block-copolymer. For instance, the
monolayer may be composed of a poly(amino acid) such as polylysine.
A monolayer which is a self-assembled monolayer, however, is most
preferred. One face of the self-assembled monolayer is typically
composed of chemical functionalities on the termini of the organic
molecules that are chemisorbed or physisorbed onto the surface of
the substrate or, if present, the coating on the substrate if
present. Examples of suitable functionalities of monolayers include
the positively charged amino groups of poly-L-lysine for use on
negatively charged surfaces and thiols for use on gold surfaces.
Typically, the other face of the self-assembled monolayer is
exposed and may bear any number of chemical functionalities (end
groups). Preferably, the molecules of the self-assembled monolayer
are highly ordered.
A "self-assembled monolayer" is a monolayer which is created by the
spontaneous assembly of molecules. The self-assembled monolayer may
be ordered, disordered, or exhibit short- to long-range order.
An "affinity tag" is a functional moiety capable of directly or
indirectly immobilizing a protein-capture agent onto an exposed
functionality of the organic thinfilm. Preferably, the affinity tag
enables the site-specific immobilization and thus enhances
orientation of the protein-capture agent onto the organic thinfilm.
In some cases, the affinity tag may be a simple chemical functional
group. Other possibilities include amino acids, poly(amino acid)
tags, or full-length proteins. Still other possibilities include
carbohydrates and nucleic acids. For instance, the affinity tag may
be a polynucleotide which hybridizes to another polynucleotide
serving as a functional group on the organic thinfilm or another
polynucleotide serving as an adaptor. The affinity tag may also be
a synthetic chemical moiety. If the organic thinfilm of each of the
patches comprises a lipid bilayer or monolayer, then a membrane
anchor is a suitable affinity tag. The affinity tag may be
covalently or noncovalently attached to the protein-capture agent.
For instance, if the affinity tag is covalently attached to the
protein-capture agent it may be attached via chemical conjugation
or as a fusion protein. The affinity tag may also be attached to
the protein-capture agent via a cleavable linkage. Alternatively,
the affinity tag may not be directly in contact with the
protein-capture agent. The affinity tag may instead be separated
from the protein-capture agent by an adaptor. The affinity tag may
immobilize the protein-capture agent to the organic thinfilm either
through noncovalent interactions or through a covalent linkage.
An "adaptor", for purposes of this invention, is any entity that
links an affinity tag to the protein-capture agent. The adaptor may
be, but need not necessarily be, a discrete. molecule that is
noncovalently attached to both the affinity tag and the
protein-capture agent. The adaptor can instead be covalently
attached to the affinity tag or the protein-capture agent or both
(via chemical conjugation or as a fusion protein, for instance).
Proteins such as full-length proteins, polypeptides, or peptides
are typical adaptors. Other possible adaptors include carbohydrates
or nucleic acids.
The term "fusion protein" refers to a protein composed of two or
more polypeptides that, although typically unjoined in their native
state, are joined by their respective amino and carboxyl termini
through a peptide linkage to form a single continuous polypeptide.
It is understood that the two or more polypeptide components can
either be directly joined or indirectly joined through a peptide
linker/spacer.
The term "normal physiological condition" means conditions that are
typical inside a living organism or a cell. While it is recognized
that some organs or organisms provide extreme conditions, the
intra-organismal and intra-cellular environment normally varies
around pH 7 (i.e., from pH 6.5 to pH 7.5), contains water as the
predominant solvent, and exists at a temperature above 0.degree. C.
and below 50.degree. C. It will be recognized that the
concentration of various salts depends on the organ, organism,
cell, or cellular compartment used as a reference.
(b) Arrays of the Invention
The present invention is directed to arrays of protein-capture
agents which can bind a plurality of proteins that are the
expression products, or fragments thereof, of a cell or population
of cells in an organism and therefore can be used to evaluate gene
expression at the protein level. Typically, the arrays comprise
micrometer-scale, two-dimensional patterns of patches of
protein-capture agents immobilized on an organic thinfilm coating
on the surface of the substrate.
In one embodiment of the invention, the array of protein-capture
agents comprises a substrate, at least one organic thinfilm
covering some or all of the surface of the substrate, and a
plurality of patches arranged in discrete, known regions on the
portions of the substrate surface covered by organic thinfilm,
wherein (i) each patch comprises protein-capture agents immobilized
on the organic thinfilm, wherein said protein-capture agents of a
given patch are capable of binding a particular expression product,
or a fragment thereof, of a cell or population of cells in an
organism, and (ii) the array comprises a plurality of different
protein-capture agents, each of which is capable of binding a
different expression product, or fragment thereof, of the cell or
population of cells.
The protein-capture agents are preferably covalently immobilized on
the patches of the array; either directly or indirectly.
In most cases, the array will comprise at least about ten patches.
In a preferred embodiment, the array comprises at least about 50
patches. In a particularly preferred embodiment the array comprises
at least about 100 patches. In alternative preferred embodiments,
the array of protein-capture agents may comprise more than
10.sup.3, 10.sup.4 or 10.sup.5 patches.
The area of surface of the substrate covered by each of the patches
is preferably no more than about 0.25 mm.sup.2. Preferably, the
area of the substrate surface covered by each of the patches is
between about 1 .mu.m.sup.2 and about 10,000 .mu.m.sup.2. In a
particularly preferred embodiment, each patch covers an area of the
substrate surface from about 100 .mu.m.sup.2 to about 2,500
.mu.m.sup.2. In an alternative embodiment, a patch on the array may
cover an area of the substrate surface as small as about 2,500
nm.sup.2, although patches of such small size are generally not
necessary for the use of the array.
The patches of the array may be of any geometric shape. For
instance, the patches may be rectangular or circular. The patches
of the array may also be irregularly shaped. The patches are
optionally elevated from the median plan of the underlying
substrate.
The distance separating the patches of the array can vary.
Preferably, the patches of the array are separated from neighboring
patches by about 1 .mu.m to about 500 .mu.m. Typically, the
distance separating the patches is roughly proportional to the
diameter or side length of the patches on the array if the patches
have dimensions greater than about 10 .mu.m. If the patch size is
smaller, then the distance separating the patches will typically be
larger than the dimensions of the patch.
In a preferred embodiment of the array, the patches of the array
are all contained within an area of about 1 cm.sup.2 or less on the
surface of the substrate. In one preferred embodiment of the array,
therefore, the array comprises 100 or more patches within a total
area of about 1 cm.sup.2 or less on the surface of the substrate.
Alternatively, a particularly preferred array comprises 10.sup.3 or
more patches within a total area of about 1 cm.sup.2 or less. A
preferred array may even optionally comprise 10.sup.4 or 10.sup.5
or more patch within an area of about 1 cm.sup.2 or less on the
surface of the substrate. In other embodiments of the invention,
all of the patches of the array are contained within an area of
about 1 mm.sup.2 or less on the surface of the substrate.
Typically, only one type of protein-capture agent is present on a
single patch of the array. If more than one type of protein-capture
agent is present on a single patch, all of the protein-capture
agents of that patch must share a common binding partner. For
instance, a patch may comprise a variety of polyclonal antibodies
to the same antigen (although, potentially, the antibodies may bind
different epitopes on that same antigen).
The arrays of the invention can have any number of a plurality of
different protein-capture agents. Typically the array comprises at
least about ten different protein-capture agents. Preferably, the
array comprises at least about 50 different protein-capture agents.
More preferably, the array comprises at least about 100 different
protein-capture agents. Alternative preferred arrays comprise more
than about 10.sup.3 different protein-capture agents or more than
about 10.sup.4 different protein-capture agents. The array may even
optionally comprise more than about 10.sup.5 different
protein-capture agents.
The number of different protein-capture agents on the array will
vary depending if on the application desired. For instance, if the
array is to be used as a diagnostic tool in evaluating the status
of a tumor or other diseased tissue in a patient, an array
comprising less than about 100 different protein-capture agents may
suffice since the necessary binding partners of the protein-capture
agent on the array are limited to only those proteins whose
expression is known to be indicative of the disease condition.
However, if the array is to be used to measure a significant
portion of the total protein content of a cell, then the array
preferably comprises at least about 10,000 different
protein-capture agents. Alternatively, a more limited proteomics
study, such as a study of the abundances of various human
transcription factors, for instance, might only require an array of
about 100 different protein-capture agents.
In one embodiment of the array, each of the patches of the array
comprises a different protein-capture agent. For instance, an array
comprising about 100 patches could comprise about 100 different
protein-capture agents. Likewise, an array of about 10,000 patches
could comprise about 10,000 different protein-capture agents. In an
alternative embodiment, however, each different protein-capture
agent is immobilized on more than one separate patch on the array.
For instance, each different protein-capture agent may optionally
be present on two to six different patches. An array of the
invention, therefore, may comprise about three-thousand
protein-capture agent patches, but only comprise about one thousand
different protein-capture agents since each different
protein-capture agent is present on three different patches.
Typically, the number of different proteins which can be bound by
the plurality of different protein-capture agents on the array will
be at least about ten. However, it is preferred that the plurality
of different protein-capture agents on the array is capable of
binding a higher number of different proteins, such as at least
about 50 or at least about 100. In still further preferred
embodiments, the plurality of different proteins on the array is
capable of binding at least about 10.sup.3 proteins. For some
applications, such as those where it is desirable to assay the
entire protein content of a cell, or a significant fraction
thereof, an array where the plurality of protein-capture agents is
capable of binding at least about 10.sup.4 different proteins or
even at least about 10.sup.5 different proteins is most
preferred.
In one embodiment of the invention, the binding partners of the
plurality of protein-capture agents on the array are proteins which
are all expression products, or fragments thereof, of a cell or
population of cells of a single organism. The expression products
may be proteins, including peptides, of any size or function. They
may be intracellular proteins or extracellular proteins. The
expression products may be from a one-celled or multicellular
organism. The organism may be a plant or an animal. In a preferred
embodiment of the invention, the binding partners are human
expression products, or fragments thereof.
In one embodiment of the invention, the binding partners of the
protein-capture agents of the array may be a randomly chosen subset
of all the proteins, including peptides, which are expressed by a
cell or population of cells in a given organism or a subset of all
the fragments of those proteins. Thus, the binding partners of the
protein-capture agents of the array optionally represent a wide
distribution of different proteins from a single organism.
The binding partners of some or all of the protein-capture agents
on the array need not necessarily be known. The binding partner of
a protein-capture agent of the array may be a protein or peptide of
unknown function. For instance, the different protein-capture
agents of the array may together bind a wide range of cellular
proteins from a single cell type, many of which are of unknown
identity and/or function.
In another embodiment of the present invention, the binding
partners of the protein-capture agents on the array are related
proteins. The different proteins bound by the protein-capture
agents may optionally be members of the same protein family. The
different binding partners of the protein-capture agents of the
array may be either functionally related or just suspected of being
functionally related. The different proteins bound by the
protein-capture agents of the array may also be proteins which
share a similarity in structure or sequence or are simply suspected
of sharing a similarity in structure or sequence. For instance, the
binding partners of the protein-capture agents on the array may
optionally all be growth factor receptors, hormone receptors,
neurotransmitter receptors, catecholamine receptors, amino acid
derivative receptors, cytokine receptors, extracellular matrix
receptors, antibodies, lectins, cytokines, serpins, proteases,
kinases, phosphatases, ras-like GTPases, hydrolases, steroid
hormone receptors, transcription factors, heat-shock transcription
factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper
proteins, homeodomain proteins, intracellular signal transduction
modulators and effectors, apoptosis-related factors, DNA synthesis
factors DNA repair factors, DNA recombination factors, cell-surface
antigens, hepatitis C virus (HCV) proteases or HIV proteases.
In an alternative embodiment of the invention, the proteins which
are the binding partners of the protein-capture agents of the array
may be fragments of the expression products of a cell or population
of cells in an organism.
A protein-capture agent on the array can be any molecule or complex
of molecules which has the ability to bind a protein and immobilize
it to the site of the protein-capture agent on the array.
Preferably, the protein-capture agent binds its binding partner in
a substantially specific manner. Hence, the protein-capture agent
may optionally be a protein whose natural function in a cell is to
specifically bind another protein, such as an antibody or a
receptor. Alternatively, the protein-capture agent may instead be a
partially or wholly synthetic or recombinant protein which
specifically binds a protein. Alternatively, the protein-capture
agent may be a protein which has been selected in vitro from a
mutagenized, randomized, or completely random and synthetic library
by its binding affinity to a specific protein or peptide target.
The selection method used may optionally have been a display method
such as ribosome display or phage display (see below).
Alternatively, the protein-capture agent obtained via in vitro
selection may be a DNA or RNA aptamer which specifically binds a
protein target (for example: Potyrailo et al., Anal. Chem.,
70:3419-25, 1998; Cohen, et al., Proc. Natl. Acad. Sci. USA,
95:14272-7, 1998; Fukuda, et al., Nucleic Acids Symp. Ser.,
(37):237-8, 1997). Alternatively, the in vitro selected
protein-capture agent may be a polypeptide (Roberts and Szostak,
Proc. Natl. Acad. Sci. USA, 94:12297-302, 1997). In an alternative
embodiment, the protein-capture agent may be a small molecule which
has been selected from a combinatorial chemistry library or is
isolated from an organism.
In a preferred embodiment of the array, however, the
protein-capture agents are proteins. In a particularly preferred
embodiment, the protein-capture agents are antibodies or antibody
fragments. Although antibody moieties are exemplified herein, it is
understood that the present arrays and methods may be
advantageously employed with other protein-capture agents.
The antibodies or antibody fragments of the array may optionally be
single-chain Fvs, Fab fragments, Fab' fragments, F(ab').sub.2
fragments, Fv fragments, dsFvs diabodies, Fd fragments,
fill-length, antigen-specific polyclonal antibodies, or full-length
monoclonal antibodies. In a preferred embodiment, the
protein-capture agents of the array are monoclonal antibodies, Fab
fragments or single-chain Fvs.
The antibodies or antibody fragments may be monoclonal antibodies,
even commercially available antibodies, against known,
well-characterized proteins. Alternatively, the antibody fragments
have been derived by selection from a library using the phage
display method. If the antibody fragments are derived individually
by selection based on binding affinity to known proteins, then, the
binding partners of the antibody fragments are known. In an
alternative embodiment of the invention, the antibody fragments
have been derived by a phage display method comprising selection
based on binding affinity to the (typically, immobilized) proteins
of a cellular extract or a body fluid. In this embodiment, some or
many of the antibody fragments of the array would bind proteins of
unknown identity and/or function.
Upon using the array of protein-capture agents to bind a plurality
of expression products, or fragments thereof, an array of bound
proteins is created. Thus, another embodiment of the invention
provides an array of bound proteins which comprises (a) a
protein-capture agent array of the invention and (b) a plurality of
different proteins which are expression products, or fragments
thereof, of a cell or a population of cells in an organism, wherein
each of the different proteins is bound to a protein-capture agent
on a separate patch of the array. Preferably, each of the different
proteins is non-covalently bound to a protein-capture agent.
(c) Substrates, Coatings, and Organic Thinfilms
The substrate of the array may be either organic or inorganic,
biological or non-biological, or any combination of these
materials. In one embodiment, the substrate is transparent or
translucent. The portion of the surface of the substrate on which
the patches reside is preferably flat and firm or semi-firm.
However, the array of the present invention need not necessarily be
flat or entirely two-dimensional. Significant topological features
may be present on the surface of the substrate surrounding the
patches, between the patches or beneath the patches. For instance,
walls or other barriers may separate the patches of the array.
Numerous materials are suitable for use as a substrate in the array
embodiment of the invention. For instance, the substrate of the
invention array can comprise a material selected from a group
consisting of silicon, silica, quartz, glass, controlled pore
glass, carbon, alumina, titania, tantalum oxide, germanium, silicon
nitride, zeolites, and gallium arsenide. Many metals such as gold,
platinum, aluminum, copper, titanium, and their alloys are also
options for substrates of the array. In addition, many ceramics and
polymers may also be used as substrates. Polymers which may be used
as substrates include, but are not limited to, the following:
polystyrene; poly(tetra)fluoroethylene (PTFE);
polyvinylidenedifluoride; polycarbonate; polymethylmethacrylate;
polyvinylethylene; polyethyleneimine; poly(etherether)ketone;
polyoxymethylene (POM); polyvinylphenol; polylactides;
polymethacrylimide (PMI); polyalkenesulfone (PAS);
polypropylethylene, polyethylene; polyhydroxyethylmethacrylate
(HEMA); polydimethylsiloxane; polyacrylamide; polyimide; and
block-copolymers. Preferred substrates for the array include
silicon, silica, glass, and polymers. The substrate on which the
patches reside may also be a combination of any of the
aforementioned substrate materials.
An array of the present invention may optionally further comprise a
coating between the substrate and the organic thinfilm of its
patches. This coating may either be formed on the substrate or
applied to the substrate. The substrate can be modified with a
coating by using thin-film technology based, for instance, on
physical vapor deposition (PVD), plasma-enhanced chemical vapor
deposition (PECVD), or thermal processing. Alternatively, plasma
exposure can be used to directly activate or alter the substrate
and create a coating. For instance, plasma etch procedures can be
used to oxidize a polymeric surface (for example, polystyrene or
polyethylene to expose polar functionalities such as hydroxyls,
carboxylic acids, aldehydes and the like) which then acts as a
coating.
The coating is optionally a metal film. Possible metal films
include aluminum, chromium, titanium, tantalum, nickel, stainless
steel, zinc, lead, iron, copper, magnesium, manganese, cadmium,
tungsten, cobalt, and alloys or oxides thereof. In a preferred
embodiment, the metal film is a noble metal film. Noble metals that
may be used for a coating include, but are not limited to, gold,
platinum, silver, and copper. In an especially preferred
embodiment, the coating comprises gold or a gold alloy.
Electron-beam evaporation may be used to provide a tin coating of
gold on the surface of the substrate. In a preferred embodiment,
the metal film is from about 50 nm to about 500 nm in thickness. In
an alternative embodiment, the metal film is from about 1 nm to
about 1 .mu.m in thickness.
In alternative embodiments, the coating comprises a composition
selected from the group consisting of silicon, silicon oxide,
titania, tantalum oxide, silicon nitride, silicon hydride, indium
tin oxide, magnesium oxide, alumina, glass, hydroxylated surfaces,
and polymers.
In one embodiment of the invention array, the surface of the
coating is atomically flat. In this embodiment, the mean roughness
of the surface of the coating is less than about 5 angstroms for
areas of at least 25 .mu.m.sup.2. In a preferred embodiment, the
mean roughness of the surface of the coating is less than about 3
angstroms for areas of at least 25 .mu.m.sup.2. The ultraflat
coating can optionally be a template-stripped surface as described
in Hegner et al., Surface Science, 1993, 291:39-46 and Wagner el
al., Langmuir, 1995, 11:3867-3875, both of which are incorporated
herein by reference.
It is contemplated that the coatings of many arrays will require
the addition of at least one adhesion layer between said coating
and the substrate. Typically, the adhesion layer will be at least 6
angstroms thick and may be much thicker. For instance, a layer of
titanium or chromium may be desirable between a silicon wafer and a
gold coating. In an alternative embodiment, an epoxy glue such as
Epo-tek 377.RTM., Epo-tek 301-2.RTM., (Epoxy Technology Inc.,
Billerica, Mass.) may be preferred to aid adherence of the coating
to the substrate. Determinations as to what material should be used
for the adhesion layer would be obvious to one skilled in the art
once materials are chosen for both the substrate and coating. In
other embodiments, additional adhesion mediators or interlayers may
be necessary to improve the optical properties of the array, for
instance, waveguides for detection purposes.
Deposition or formation of the coating (if present) on the
substrate is performed prior to the formation of the organic
thinfilm thereon. Several different types of coating may be
combined on the surface. The coating may cover the whole surface of
the substrate or only parts of it. The pattern of the coating may
or may not be identical to the pattern of organic thinfilm used to
immobilize the protein-capture agents. In one embodiment of the
invention, the coating covers the substrate surface only at the
site of the patches of protein-capture agents. Techniques useful
for the formation of coated patches on the surface of the substrate
which are organic thinfilm compatible are well known to those of
ordinary skill in the art. For instance, the patches of coatings on
the substrate may optionally be fabricated by photolithography,
micromolding (PCT Publication WO 96/29629), wet chemical or dry
etching, or any combination of these.
The organic thinfilm on which each of the patches of
protein-capture agents resides forms a layer either on the
substrate itself or on a coating covering the substrate. The
organic thinfilm on which the protein-capture agents of the patches
are immobilized is preferably less than about 20 nm thick. In some
embodiments of the invention, the organic thinfilm of each of the
patches may be less than about 10 nm thick.
A variety of different organic thinfilm are suitable for use in the
present invention. Methods for the formation of organic thinfilms
include in situ growth from the surface, deposition by
physisorption, spin-coating, chemisorption, self-assembly, or
plasma-initiated polymerization from gas phase. For instance, a
hydrogel composed of a material such as dextran can serve as a
suitable organic thinfilm on the patches of the array. In one
preferred embodiment of the invention, the organic thinfilm is a
lipid bilayer. In another preferred embodiment, the organic
thinfilm of each of the patches of the array is a monolayer. A
monolayer of polyarginine or polylysine adsorbed on a negatively
charged substrate or coating is one option for the organic
thinfilm. Another option is a disordered monolayer of tethered
polymer chains. In a particularly preferred embodiment the organic
thinfilm is a self-assembled monolayer. The organic thinfilm is
most preferably a self-assembled monolayer which comprises
molecules of the formula X--R--Y, wherein R is a spacer, X is a
functional group that binds R to the surface, and Y is a functional
group for binding protein-capture agents onto the monolayer. In an
alternative preferred embodiment, the self-assembled monolayer is
comprised of molecules of the formula (X).sub.a R(Y).sub.b where a
and b are, independently, integers greater than or equal to 1 and
X, R, and Y are as previously defined. In an alternative preferred
embodiment, the organic thinfilm comprises a combination of organic
thinfilm such as a combination of a lipid bilayer immobilized on
top of a self-assembled monolayer of molecules of the formula
X--R--Y. As another example, a monolayer of polylysine can also
optionally be combined with a self-assembled monolayer of molecules
of the formula X--R--Y (see U.S. Pat. No. 5,629,213).
In all cases, the coating, or the substrate itself if no coating is
present, must be compatible with the chemical or physical
adsorption of the organic thinfilm on its surface. For instance, if
the patches comprise a coating between the substrate and a
monolayer of molecules of the formula X--R--Y, then it is
understood that the coating must be composed of a material for
which a suitable functional group X is available (see below). If no
such coating is present, then it is understood that the substrate
must be composed of a material for which a suitable functional
group X is available.
In a preferred embodiment of the invention, the regions of the
substrate surface, or coating surface, which separate the patches
of protein-capture agents are free of organic thinfilm. In an
alternative embodiment, the organic thinfilm extends beyond the
area of the substrate surface, or coating surface if present,
covered by the patches of protein-capture agents. For instance,
optionally, the entire surface of the array may be covered by an
organic thinfilm on which the plurality of spatially distinct
patches of protein-capture agents reside. An organic thinfilm which
covers the entire surface of the array may be homogenous or may
optionally comprise patches of differing exposed functionalities
useful in the immobilization of patches of different
protein-capture agents. In still another alternative embodiment,
the regions of the substrate surface or coating surface, if a
coating is present, between the patches of protein-capture agents
are covered by an organic thinfilm, but an organic thinfilm of a
different type than that of the patches of protein-capture agents.
For instance, the surfaces between the patches of protein-capture
agents may be coated with an organic thinfilm characterized by low
non-specific binding properties for proteins and other
analytes.
A variety of techniques may be used to generate patches of organic
thinfilm on the surface of the substrate or on the surface of a
coating on the substrate. These techniques are well known to those
skilled in the art and will vary depending upon the nature of the
organic thinfilm, the substrate, and the coating if present. The
techniques will also vary depending on the structure of the
underlying substrate and the pattern of any coating a present on
the substrate. For instance, patches of a coating which is highly
reactive with an organic thinfilm may have already been produced on
the substrate surface. Arrays of patches of organic thinfilm can
optionally be created by microfluidics printing, microstamping
(U.S. Pat. Nos. 5,512,131 and 5,731,152), or microcontact printing
(.mu.CP) (PCT Publication WO 96/29629). Subsequent immobilization
of protein-capture agents to the reactive monolayer patches results
in two-dimensional arrays of the agents. Inkjet printer heads
provide another option for patterning monolayer X--R--Y molecules,
or components thereof, or other organic thinfilm components to
nanometer or micrometer scale sites on the surface of the substrate
or coating (Lemmo et al., Anal Chem., 1997, 69:543-551; U.S. Pat.
Nos. 5,843,767 and 5,837,860). In some cases, commercially
available arrayers based on capillary dispensing (for instance,
OmniGrid.TM. from Genemachines, inc, San Carlos, Calif., and
High-Throughput Microarrayer from Intelligent Bio-Instrments,
Cambridge, Mass.) may also be of use in directing components of
organic thinfilis to spatially distinct regions of the array.
Diffusion boundaries between the patches of protein-capture agents
immobilized on organic thinfilms such as self-assembled monolayers
maybe integrated as topographic patterns (physical barriers) or
surface functionalities with orthogonal wetting behavior (chemical
barriers). For instance, walls of substrate material or photoresist
may be used to separate some of the patches from some of the others
or all of the patches from each other. Alternatively,
non-bioreactive organic thinfims, such as monolayers, with
different wettability may be used to separate patches from one
another.
In a preferred embodiment of the invention, each of the patches of
protein-capture agents comprises a self-assembled monolayer of
molecules of the formula X--R--Y, as previously defined, and the
patches are separated from each other by surfaces free of the
monolayer.
FIG. 1 shows the top view of one example of an array of patches
reactive with protein-capture agents. On the array, a number of
patches 15 cover the surface of the substrate 3.
FIG. 2 shows a detailed cross section of a patch 15 of the array of
FIG. 1. This view illustrates the use of a coating 5 on the
substrate 3. An adhesion interlayer 6 is also included in the
patch. On top of the patch resides a self-assembled monolayer
7.
FIG. 3 shows a cross section of one row of the patches 15 of the
array of FIG. 1. This figure also shows the use of a cover 2 over
the array. Use of the cover 2 creates an inlet port 16 and an
outlet port 17 for solutions to be passed over the array.
A variety of chemical moieties may function as monolayer molecules
of the formula X--R--Y in the array of the present invention.
However, three major classes of monolayer formation are preferably
used to expose high densities of reactive omega-functionalities on
the patches of the array: (i) alkylsiloxane monolayers ("silanes")
on hydroxylated and non-hydroxylated surfaces (as taught in, for
example, U.S. Pat. No. 5,405,766, PCT Publication WO 96/38726, U.S.
Pat. No. 5,412,087, and U.S. Pat. No. 5,688,642); (ii)
alkyl-thiol/dialkyldisulfide monolayers on noble metals (preferably
Au(111)) (as, for example, described in Allara et al., U.S. Pat.
No. 4,690,715; Bamdad et al., U.S. Pat. No. 5,620,850; Wagner et
al., Biophysical Journal, 1996, 70:2052-2066); and (iii) alkyl
monolayer formation on oxide-free passivated silicon (as taught in,
for example, Linford et al., J. Am. Chem. Soc., 1995,
117:3145-3155, Wagner et al., Journal of structural Biology, 1997,
119:189-201, U.S. Pat. No. 5,429,708). One of ordinary skill in the
art, however, will recognize that many possible moieties may be
substituted for X, R, and/or Y, dependent primarily upon the choice
of substrate, coating, and affinity tag. Many examples of
monolayers are described in Ulman, An Introduction to Ultrathin
Organic Films: From Langmuir-Blodgett to SelfAssembly, Academic
press (1991).
In one embodiment, the monolayer comprises molecules of the formula
(X).sub.a R(Y).sub.b wherein a and b are, independently, equal to
an integer between 1 and about 200. In a preferred embodiment, a
and b are, independently, equal to an integer between 1 and about
80. In a more preferred embodiment, a and b are, independently,
equal to 1 or 2. In a most preferred embodiment, a and b are both
equal to 1 (molecules of the formula X--R--Y).
If the patches of the invention array comprise a self-assembled
monolayer of molecules of the formula (X).sub.a R(Y).sub.b, then R
may optionally comprise a linear or branched hydrocarbon chain from
about 1 to about 400 carbons long. The hydrocarbon chain may
comprise an alkyl, aryl, alkenyl, alkynyl, cycloalkyl, alkaryl,
aralkyl group, or any combination thereof. If a and b are both
equal to one, then R is typically an alkyl chain from about 3 to
about 30 carbons long. In a preferred embodiment, if a and b are
both equal to one, then R is an alkyyl chain from about 8 to about
22 carbons long and is, optionally, a straight alkane. However, it
is also contemplated that in an alternative embodiment, R may
readily comprise a linear or branched hydrocarbon chain from about
2 to about 400 carbons long and be interrupted by at least one
hetero atom. The interrupting hetero groups can include --O--,
--CONH--, --CONHCO--, --NH--, --CSNH--, --CO--, --CS--, --S--,
--SO--, --(OCH.sub.2 CH.sub.2).sub.n -- (where n=1-20),
--(CF.sub.2).sub.n -- (where n=1-22), and the like. Alternatively,
one or more of the hydrogen moieties of R can be substituted with
deuterium. In alternative, less preferred, embodiments, R may be
more than about 400 carbons long.
X may be chosen as any group which affords chemisorption or
physisorption of the monolayer onto the surface of the substrate
(or the coating, if present). When the substrate or coating is a
metal or metal alloy, X, at least prior to incorporation into the
monolayer, can in one embodiment be chosen to be an asymmetrical or
symmetrical disulfide, sulfide, diselenide, selenide, thiol,
isonitrile, selenol, a trivalent phosphorus compound,
isothiocyanate, isocyanate, xanthanate, thiocarbamate,a phosphine,
an amine, thio acid or a ditlio acid. This embodiment is especially
preferred when a coating or substrate is used that is a noble metal
such as gold, silver, or platinumn.
If the substrate of the array is a material such as silicon,
silicon oxide, indium tin oxide, magnesium oxide, alumina, quartz,
glass, or silica, then the array of one embodiment of the invention
comprises an X that, prior to incorporation into said monolayer, is
a monohalosilane, dihalosilane, trihalosilane, trialkoxysilane,
Addialkoxysilane, or a monoalkoxysilane. Among these silanes,
trichlorosilane and trialkoxysilane are particularly preferred.
In a preferred embodiment of the invention, the substrate is
selected from the group consisting of silicon, silicon dioxide,
indium tin oxide, alumina, glass, and titania; and X, prior to
incorporation into said monolayer, is selected from the group
consisting of a monohalosilane, dihalosilane, trihalosilane,
trichlorosilane, trialkoxysilane, dialkoxysilane, monoalkoxysilane,
carboxylic acids, and phosphates.
In another preferred embodiment of the invention, the substrate of
the array is silicon and X is an olefin.
In still another preferred embodiment of the invention, the coating
(or the substrate if no coating is present) is titania or tantalum
oxide and X is a phosphate.
In other embodiments, the surface of the substrate (or coating
thereon) is composed of a material such as titanium oxide, tantalum
oxide, indium tin oxide, magnesium oxide, or alumina where X is a
carboxylic acid or alkylphosphoric acid. Alternatively, if the
surface of the substrate (or coating thereon) of the array is
copper, then X may optionally be a hydroxamic acid.
If the substrate used in the invention is a polymer, then in many
cases a coating on the substrate such as a copper coating will be
included in the array. An appropriate functional group X for the
coating would then be chosen for use in the array. In an
alternative embodiment comprising a polymer substrate, the surface
of the polymer may be plasma-modified to expose desirable surface
functionalities for monolayer formation. For instance, EP 780423
describes the use of a monolayer molecule that has an alkene X
functionality on a plasma exposed surface. Still another
possibility for the invention array comprised of a polymer is that
the surface of the polymer on which the monolayer is formed is
functionality by copolymerization of appropriately functionality
precursor molecules.
Another possibility is that prior to incorporation into the
monolayer, X can be a free-radical-producing moiety. This
functional group is especially appropriate when the surface on
which the monolayer is formed is a hydrogenated silicon surface.
Possible free-radical producing moieties include, but are not
limited to, diacylperoxides, peroxides, and azo compounds.
Alternatively, unsaturated moieties such as unsubstituted alkenes,
alkynes, cyano compounds and isonitrile compounds can be used for X
if the reaction with X is accompanied by ultraviolet, infrared,
visible, or microwave radiation.
In alternative embodiments, X, prior to incorporation into the
monolayer, may be a hydroxyl, carboxyl, vinyl, sulfonyl,
phosphoryl, silicon hydride, or an amino group.
The component, Y, of the monolayer is a functional group
responsible for binding a protein-capture agent onto the monolayer.
In a preferred embodiment of the invention, the Y group is either
highly reactive (activated) towards the protein-capture agent (or
its affinity tag) or is easily converted into such an activated
form. In a preferred embodiment, the coupling of Y with the
protein-capture agent occurs readily under normal physiological
conditions not detrimental to the ability of the protein-capture
agent to bind its binding partner. The functional group Y may
either form a covalent linkage or en a noncovalent linkage with the
protein-capture agent (or its affinity tag, if present). In a
preferred embodiment, the functional group Y forms a covalent
linkage with the protein-capture agent or its affinity tag. It is
understood that following the attachment of the protein-capture
agent (with or without an affinity tag) to Y, the chemical nature
of Y may have changed. Upon attachment of the protein-capture
agent, Y may even have been removed from the organic thinfilm.
In one embodiment of the array of the present invention, Y is a
functional group that is activated in situ. Possibilities for this
type of functional group include, but are not limited to, such
simple moieties such as a hydroxyl, carboxyl, amino, aldehyde,
carbonyl, methyl, methylene, alkene, alkyne, carbonate, aryliodide,
or a vinyl group. Appropriate modes of activation would be obvious
to one skilled in the art. Alternatively, Y can comprise a
functional group that requires photoactivation prior to becoming
activated enough to trap the protein-capture agent.
In an especially preferred embodiment of the array of the present
invention, Y is a complex and highly reactive functional moiety
that is compatible with monolayer formation and needs no in situ
activation prior to reaction with the protein-capture agent and/or
affinity tag. Such possibilities for Y include, but are not limited
to, maleimide, N-hydroxysuccinimide (Wagner et al., Biophysical
Journal, 1996, 70:2052-2066), nitrilotriacetic acid (U.S. Pat. No.
5,620,850), activated hydroxyl, haloacetyl, bromoacetyl,
iodoacetyl, activated carboxyl, hydrazide, epoxy, aziridine,
sulfonylchloride, trifiuoromethyldiaziridine, pyridyldisulfide,
N-acyl-imidazole, imidazolecarbamate, vinylsulfone,
succinimidylcarbonate, arylazide, anhydride, diazoacetate,
benzophenone, isothiocyanate, isocyanate, imidoester,
fluorobenzene, and biotin.
FIG. 4 shows one example of a monolayer on a substrate 3. In this
example, substrate 3 comprises glass. The monolayer is
thiolreactive because it bears a maleimidyl functional group Y.
FIG. 5 shows another example of a monolayer on a substrate 3 which
is silicon. In this case, however, a thinfilm gold coating 5 covers
the surface of the substrate 3. Also, in this embodiment, a
titanium adhesion interlayer 6 is used to adhere the coating 5 to
the substrate 3. This monolayer is aminoreactive because it bears
an N-hydroxysuccinimdyl functional group Y.
In an alternative embodiment, the functional group Y of the array
is selected from the group of simple functional moieties. Possible
Y functional groups include, but are not limited to, --OH,
--NH.sub.2, --COOH, --COOR, --RSR, --PO.sub.4.sup.-3,
--OSO.sub.3.sup.-2, --SO.sub.3.sup.-, --COO.sup.-, --SOO.sup.-,
--CONR.sub.2, --CN, --NR.sub.2, and the like.
The monolayer molecules of the present invention can optionally be
assembled on the surface in parts. In other words, the monolayer
need not necessarily be constructed by chemisorption or
physisorption of molecules of the formula X--R--Y to the surface of
the substrate (or coating). Instead, in one embodiment, X may be
chemisorbed or physisorbed to the surface of the substrate (or
coating) alone first. Then, R or even just individual components of
R can be attached to X through a suitable chemical reaction. Upon
completion of addition of the spacer R to the X moiety already
immobilized on the go surface, Y can be attached to the ends of the
monolayer molecule through a suitable covalent linkage.
Not all self-assembled monolayer molecules on a given patch need be
identical to one another. Some patches may comprise mixed
monolayers. For instance, the monolayer of an individual patch may
optionally comprise at least two different molecules of the formula
X--R--Y, as previously described. This second X--R--Y molecule may
immobilize the same or a different protein-capture agent having the
same binding partner as the first. In addition, some of the
monolayer molecules X--R--Y of a patch may have failed to attach
any protein-capture agent.
As another alternative embodiment of the invention, a mixed,
self-assembled monolayer of an individual patch on the array may
comprise both molecules of the formula X--R--Y, as previously
described, and molecules of the formula, X--R--V where R is a
spacer, X is a functional group that binds R to the surface, and V
is a moiety which is biocompatible with proteins and resistant to
the non-specific binding of proteins. For example, V may consist of
a hydroxyl, saccharide, or oligo/polyethylene glycol moiety (EP
Publication 780423).
In still another embodiment of the invention, the array comprises
at least one unreactive patch of organic thinfilm on the substrate
or coating surface which is devoid of any protein-capture agent.
For instance, the unreactive patch may optionally comprise a
monolayer of molecules of the formula X--R--V, where R is a spacer,
X is a functional group that binds R to the surface, and V is a
moiety resistant to the non-specific binding of proteins. The
unreactive patch may serve as a control patch or be useful in
background binding measurements.
Regardless of the nature of the monolayer molecules, in some arrays
it may be desirable to provide crosslinking between molecules of an
individual patch's monolayer. In general, crosslinking confers
additional stability to the monolayer. Such methods are familiar to
those skilled in the art (for instance, see Ulman, An Introduction
to Ultrathin Organic Films: From Langmuir-Blodgett to
Self-Assembly, Academic Press (1991)).
After completion of formation of the monolayer on the patches, the
protein-capture agent may be attached to the monolayer via
interaction with the Y-functional group. Y-functional groups which
fail to react with any protein-capture agents are preferably
quenched prior to use of the array.
(d) Affinity Tags and Immobilization of Protein-capture Agents
In a preferred embodiment, the protein-immobilizing patches of the
array further comprise an affinity tag that enhances,
immobilization of the protein-capture agent onto the organic
thinfilm. The use of an affinity tag on the protein-capture agent
of the array typically provides several advantages. An affinity tag
can confer enhanced binding or reaction of the protein-capture
agent with the functionalities on the organic thinfilm, such as Y
if the organic thinfilm is a an X--R--Y monolayer as previously
described. This enhancement effect may be either kinetic or
thermodynamic. The affinity tag/thinfilm combination used in the
patches of the array preferably allows for immobilization of the
protein-capture agents in a manner which does not require harsh
reaction conditions that are adverse to protein stability or
function. In most embodiments, immobilization to the organic
thinfilm in aqueous, biological buffers is ideal.
An affinity tag also preferably offers immobilization on the
organic thinfilm that is specific to a designated site or location
on the protein-capture agent (site-specific immobilization). For
this to occur, attachment of the affinity tag to the
protein-capture agent must be site-specific. Site-specific
immobilization helps ensure that the protein-binding site of the
agent, such as the antigen-binding site of the antibody moiety,
remains accessible to ligands in solution. Another advantage of
immobilization through affinity tags is that it allows for a common
immobilization strategy to be used with multiple, different
protein-capture agents.
The affinity tag is optionally attached directly, either covalently
or noncovalently, to the protein-capture agent. In an alternative
embodiment, however, the affinity tag is either covalently or
noncovalently attached to an adaptor which is either covalendy or
noncovalently attached to the protein-capture agent.
In a preferred embodiment, the affinity tag comprises at least one
amino acid. The affinity tag may be a polypeptide comprising at
least two amino acids which is reactive with the functionalities of
the organic thinfilm. Alternatively, the affinity tag may be a
single amino acid which is reactive with the organic thinfilm.
Examples of possible amino acids which could be reactive with an
organic thinfilm include cysteine, lysine, histidine, arginine,
tyrosine, aspartic acid, glutamic acid, tryptophan, serine,
threonine, and glutamine. A polypeptide or amino acid affinity tag
is preferably expressed as a fusion protein with the
protein-capture agent when the protein-capture agent is a protein,
such as an antibody or antibody fragment. Amino acid affinity tags
provide either a single amino acid or a series of amino acids that
can interact with the functionality of the organic thinfilm, such
as the Y-functional group of the self-assembled monolayer
molecules. Amino acid affinity tags can be readily introduced into
recombinant proteins to facilitate oriented immobilization by
covalent binding to the Y-functional group of a monolayer or to a
functional group on an alternative organic thinfilm.
The affinity tag may optionally comprise a poly(amino acid) tag. A
poly(amino acid) tag is a polypeptide that comprises from about 2
to about 100 residues of a single amino acid, optionally
interrupted by residues of other amino acids. For instance, the
affinity tag may comprise a poly-cysteine, polylysine,
poly-arginine, or poly-histidine. Amino acid tags are preferably
composed of two to twenty residues of a single amino acid, such as,
for example, histidines, lysines, arginines, cysteines, glutamines,
tyrosines, or any combination of these. According to a preferred
embodiment, an amino acid tag of one to twenty amino acids includes
at least one to ten cysteines for thioether linkage; or one to ten
lysines for amide linkage; or one to ten arginines for coupling to
vicinal dicarbonyl groups. One of ordinary skill in the art can
readily pair suitable affinity tags with a given functionality on
an organic thinfilm.
The position of the amino acid tag can be at an amino-, or
carboxy-terminus of the protein-capture agent which is a protein,
or anywhere in-between, as long as the protein-binding region of
the protein-capture agent, such as the antigen-binding region of an
immobilized antibody moiety, remains in a position accessible for
protein binding. Where compatible with the protein-capture agent
chosen, affinity tags introduced for protein purification are
preferentially located at the C-terminus of the recombinant protein
to ensure that only full-length proteins are isolated during
protein purification. For instance, if intact antibodies are used
on the arrays, then the attachment point of the affinity tag on the
antibody is preferably located at a C-terminus of the effector (Fc)
region of the antibody. If scFvs are used on the arrays, then the
attachment point of the affinity tag is also preferably located at
the C-terminus of the molecules.
Affinity tags may also contain one or more unnatural amino acids.
Unnatural amino acids can be introduced using suppressor tRNAs that
recognize stop codons (i.e., amber) (Noren et al, Science, 1989,
244:182-188; Ellman et al., Methods Enzym., 1991, 202:301-336;
Cload et al., Chem. Biol., 1996, 3:1033-1038). The tRNAs are
chemically amino-acylated to contain chemically altered
("unnatural") amino acids for use with specific coupling
chemistries (i.e., ketone modifications, photoreactive groups).
In an alternative embodiment the affinity tag can comprise an
intact protein, such as, but not limited to, glutathione
S-transferase, an antibody, avidin, or streptavidin.
When the protein-capture agent is a protein and the affinity tag is
a protein, such as a poly(amino acid) tag, or a single amino acid,
the affinity tag is preferably attached to the protein-capture
agent by generating a fusion protein. Alternatively, protein
synthesis or protein ligation techniques known to those skilled in
the art may be used. For instance, intein-mediated protein ligation
may optionally be used to attach the affinity tag to the
protein-capture agent (Mathys, et al., Gene 231:1-13, 1999; Evans,
et al., Protein Science 7:2256-2264, 1998).
Other protein conjugation and immobilization techniques known in
the art may be adapted for the purpose of attaching affinity tags
to the protein-capture agent. For instance, in an alternative
embodiment of the array, the affinity tag may be an organic
bioconjugate which is chemically coupled to the protein-capture
agent of interest. Biotin or antigens may be chemically cross
linked to the protein. Alternatively, a chemical crosslinker may be
used that attaches a simple functional moiety such as a thiol or an
amine to the surface of a protein serving as a protein-capture
agent on the array.
In an alternative embodiment of the invention, the organic thinfilm
of each of the patches comprises, at least in part, a lipid
monolayer or bilayer, and the affinity tag comprises a membrane
anchor.
FIG. 6 shows a detailed cross section of a patch on one embodiment
of the invention array. In this embodiment, a protein-capture agent
10 is immobilized on a en monolayer 7 on a substrate 3. An affinity
tag 8 connects the protein-capture agent 10 to the monolayer 7. The
monolayer 7 is formed on a coating 5 which is separated from the
substrate 3 by an interlayer 6.
In an alternative embodiment of the invention, no affinity tag is
used to immobilize the protein-capture agents onto the organic
thinfilm. An amino acid or other moiety (such as a carbohydrate
moiety) inherent to the protein-capture agent itself may instead be
used to tether the protein-capture agent to the reactive group of
the organic thinfilm. In preferred embodiments, the immobilization
is site-specific with respect to the location of the site of
immobilization on the protein-capture agent. For instance, the
sulfhydryl group on the C-terminal region of the heavy chain
portion of a Fab' fragment generated by pepsin digestion of an
antibody, followed by selective reduction of the disulfide between
monovalent Fab' fragments, may be used as the affinity tag.
Alternatively, a carbohydrate moiety on the Fc portion of an intact
antibody can be oxidized under mild conditions to an aldehyde group
suitable for immobilizing the antibody on a monolayer via reaction
with a hydrazide-activated Y group on the monolayer. Examples of
immobilization of protein-capture agents without any affinity tag
in a site-specific manner can be found in Dammer et al., Biophys
J., 70:2437-2441, 1996 and the specific examples, Examples 5-7,
below.
Since the protein-capture agents of at least some of the different
patches on the array are different from each other, different
solutions, each containing a different, preferably, affinity-tagged
protein-capture agent, must be delivered to their individual
patches. Solutions of protein-capture agents may be transferred to
the appropriate patches via arrayers which are well-known in the
art and even commercially available. For instance,
microcapillary-based dispensing systems may be used. These
dispensing systems are preferably automated and computer-aided. A
description of and building instructions for an example of a
microarrayer comprising an automated capillary system can be found
on the internet at http://cmgm.stanford.edu/pbrown/array.html and
http:f//cmgm.stanford.edu/pbrown/mguide/index.html. The use of
other microprinting techniques for transferring solutions
containing the protein-capture agents to the agent-reactive patches
is also possible. Ink-jet printer heads may also optionally be used
for precise delivery of the protein-capture agents to the
agent-reactive patches. Representative, non-limiting disclosures of
techniques useful for depositing the protein-capture agents on the
patches may be found, for example, in U.S. Pat. Nos.
5,731,152(stamping apparatus), U.S. Pat. No. 5;807,522 (capillary
dispensing device), U.S. Pat. No. 5,837,860 (ink-jet printing
technique, Hamilton 2200 robotic pipetting delivery system), and
5,843,767 (ink-jet printing technique, Hamilton 2200 robotic
pipetting delivery system), all incorporated by reference
herein.
(e) Adaptors
Another embodiment of the array of the present invention comprises
an adaptor that links the affinity tag to the protein-capture agent
on the patches of the array. The additional spacing of the
protein-capture agent from the surface of the substrate (or
coating) that is afforded by the use of an adaptor is particularly
advantageous if the protein-capture agent is a protein, since
proteins are known to be prone to surface inactivation. The adaptor
may optionally afford some additional advantages as well. For
instance, the adaptor may help facilitate the attachment of the
protein-capture agent to the affinity tag. In another embodiment,
the adaptor may help facilitate the use of a particular detection
technique with the array. One of ordinary skill in the art will be
able to choose an adaptor which is appropriate for a given affinity
tag. For instance, if the affinity tag is streptavidin, then the
adaptor could be biotin that is chemically conjugated to the
protein-capture agent which is to be immobilized.
In one embodiment, the adaptor comprises a protein. In another
embodiment, the affinity tag, adaptor, and protein-capture agent
together compose a fusion protein. Such a fusion protein may be
readily expressed using standard recombinant DNA technology.
Adaptors which are proteins are especially useful to increase the
solubility of the protein-capture agent of interest and to increase
the distance between the surface of tie substrate or coating and
the protein-capture agent. Use of a protein adaptor can also be
very useful in facilitating the preparative steps of protein
purification by affinity binding prior to immobilization on the
array. Examples of possible adaptor proteins include
glutathione-S-transferase (GST), maltose-binding protein,
chitin-binding protein, thioredoxin, green-fluorescent protein
(GFP). GFP can also be used for quantification of surface binding.
In a preferred embodiment, when the protein-capture agent is an
antibody moiety comprising the Fc region, the adaptor is a
polypeptide, such as protein G, protein A, or recombinant protein
A/G (a gene fusion product secreted from a non-pathogenic form of
Bacillus which contains four Fc binding domains from protein A and
two from protein G).
FIG. 7 shows a cross section of a patch on one particular
embodiment of the invention array. The patch comprises a
protein-capture agent 10 immobilized on a monolayer 7 via both an
affinity tag 8 and an adaptor 9. The monolayer 7 rests on a coating
5. An interlayer 6 is used between the coating 5 and the substrate
3.
(f) Preparation of the Protein-capture Agents of the Array
The protein-capture agents used on the array may be produced by any
of the variety of means known to those of ordinary skill in the
art. In a preferred embodiment of the invention, the
protein-capture agents are proteins, and in an especially preferred
embodiment, the protein-capture agents are antibodies or antibody
fragments. Therefore, methods of preparing these types of possible
protein-capture agents are emphasized here.
In preparation for immobilization to the arrays of the present
invention, the antibody moiety, or any other protein-capture agent
which is a protein or polypeptide, can optionally be expressed from
recombinant DNA either in vivo or in vitro. The cDNA of the
antibody or antibody fragment or other protein-capture agent is
cloned into an expression vector (many examples of which are
commercially available) and introduced into cells of the
appropriate organism for expression. A broad range of host cells
and expression systems may be used to produce the antibodies and
antibody fragments, or other proteins, which serve as the
protein-capture agents on the array. Expression in vivo may be done
in bacteria (for example, Escherichia coli), plants (for example,
Nicotiana tabacum), lower eukaryotes (for example, Saccharonryces
cerevisiae, Saccharomyces pombe, Pichia pastoris), or higher
eukaryotes (for example, bacculovirus-infected insect cells, insect
cells, mammalian cells). For in vitro expression PCR-amplified DNA
sequences are directly used in coupled in vitro
transcription/translation systems (for instance: Escherichia coli
S30 lysates from T7 RNA polyinerase expressing, preferably
protease-deficient strains; wheat germ lysates; reticulocyte
lysates (Promega, Pharmacia, Panvera)). The choice of organism for
optimal expression depends on the extent of post-translational
modifications (i.e., glycosylation, lipid-modifications) desired.
The choice of expression system also depends on other issues, such
as whether an intact antibody is to be produced or just a fragment
of an antibody (and which fragment), since disulfide bond formation
will be affected by the choice of a host cell. One of ordinary
skill in the art Will be able to readily choose which host cell
type is most suitable for the protein-capture agent and application
desired.
DNA sequences encoding affinity tags and adaptors can be engineered
into the expression vectors such that the protein-capture agent
genes of interest can be cloned in frame either 5' or 3' of the DNA
sequence encoding the affinity tag and adaptor protein.
The expressed protein-capture agents are purified by affinity
chromatography using commercially available resins.
Preferably, production of a plurality of protein-capture agents
involves parallel processing from cloning to protein expression and
protein purification. cDNAs for the protein-capture agent of
interest will be amplified by PCR using cDNA libraries or expressed
sequence tags (EST) clones as templates. For in vivo expression of
the proteins, cDNAs can be cloned into commercial expression vctors
(Qiagen, Novagen, Clontech) and introduced into an appropriate
organism for expression (see above). For in vitro expression
PCR-amplified DNA sequences are directly used in coupled in vitro
transcription/translation systems (see above).
Escherichia coli-based protein expression is generally the method
of choice for soluble proteins that do not require extensive
post-translational modifications for activity. Extracellular or
intracellular domains of membrane proteins will be fused to protein
adaptors for expression and purification.
The entire approach can be performed using 96-well assay plates.
PCR reactions are carried out under standard conditions.
Oligonucleotide primers contain unique restriction sites for facile
cloning into the expression vectors. Alternatively, the TA cloning
system (Clontech) can be used. The expression vectors contain the
sequences for affinity tags and the protein adaptors. PCR products
are ligated into the expression vectors (under inducible promoters)
and introduced into the appropriate competent Escherichia coli
strain by calcium-dependent transformation (strains include: XL-1
blue, BL21, SG13009(lon-)). Transformed Escherichia coli cells are
plated and individual colonies transferred into 96-array blocks.
Cultures are grown to mid-log phase, induced for expression, and
cells collected by centrifugation. Cells are resuspended containing
lysozyme and the membranes broken by rapid freeze/thaw cycles, or
by sonication. Cell debris is removed by centrifugation and the
supernatants transferred to 96-tube arrays. The appropriate
affinity matrix is added, the protein-capture agent of interest is
bound and nonspecifically bound proteins are removed by repeated
washing steps using 12-96 pin suction devices and centrifugation.
Alternatively, magnetic affinity beads and filtration devices can
be used.(Qiagen). The proteins are eluted and transferred to a new
96-well array. Protein concentrations are determined and an aliquot
of each protein-capture agent is spotted onto a nitrocellulose
filter and verified by Western analysis using an antibody directed
against the affinity tag on the protein-capture agent. The purity
of each sample is assessed by SDS-PAGE and Silver staining or mass
spectrometry. The protein-capture agents are then snap-frozen and
stored at -80.degree. C.
Saccharomyces cerevisiae allows for the production of glycosylated
protein-capture agents such as antibodies or antibody fragments.
For production in Saccharomyces cerevisiae, the approach described
above for Escherichia coli can be used with slight modifications
for transformation and cell lysis. Transformation of Saccharomyces
cerevisiae is by lithium-acetate and cell lysis is either by
lyticase digestion of the cell walls followed by freeze-thaw,
sonication or glass-bead extraction. Variations of
post-translational modifications can be obtained by using different
yeast strains (i.e., Saccharomyces pombe, Pichia pastoris).
One aspect of the bacculovirus system is the array of
post-translational modifications that can be obtained, although
antibodies and other proteins produced in bacculovirus contain
carbohydrate structures very different from those produced by
mammalian cells. The bacculovirus-infected insect cell system
requires cloning of viruses, obtaining high titer stocks and
infection of liquid insect cell suspensions (cells such as SF9,
SF21).
Mammalian cell-based expression requires transfection and cloning
of cell lines. Either lymphoid or non-lymphoid cell may be used in
the preparation of antibodies and antibody fragments. Soluble
proteins such as antibodies are collected from the medium while
intracellular or membrane bound proteins require cell lysis (either
detergent solubilization, freeze-thaw). The protein-capture agents
can then be purified analogous to the procedure described for
Escherichia coli.
For in vitro translation the system of choice is Escherichia coli
lysates obtained from protease-deficient and T7 RNA polymerase
overexpressing strains. Escherichia coli lysates provide efficient
protein expression (30-50 .mu.g/ml lysate). The entire process is
carried out in 96 well arrays. Antibody genes or other
protein-capture agent genes of interest are amplified by PCR using
oligonucleotides that contain the gene-specific sequences
containing a T7 RNA polymerase promoter and binding site and a
sequence encoding the affinity tag. Alternatively, an adaptor
protein can be fused to the gene of interest by PCR. Amplified DNAs
can be directly transcribed and translated in the Escherichia coil
lysates without prior cloning for fast analysis. The antibody
fragments or other proteins are then isolated by binding to an
affinity matrix and processed as described above.
Alternative in vitro translation systems which may be used include
wheat germ extracts and reticulocyte extracts. In vitro synthesis
of membrane proteins or post-translationally modified proteins will
require reticulocyte lysates in combination with nucrosomes.
In one embodiment of the invention, the protein-capture agents on
the array are monoclonal antibodies. The production of monoclonal
antibodies against specific protein targets is routine using
standard hybridoma technology. In fact, numerous monoclonal
antibodies are available commercially. The preparation and use of
an array of monoclonal antibodies is illustrated in the specific
example, Example 8, below.
As an alternative to obtaining antibodies or antibody fragments by
cell fusion or from continuous cell lines, the antibody moieties
may be expressed in bacteriophage. Such antibody phage display
technologies are well known to those skilled in the art. The
bacteriophage expression systems allow for the random recombination
of heavy- and light-chain sequences, thereby creating a library of
antibody sequences which can be selected against the desired
antigen. The expression system can be based on bacteriophage
.lambda. or, more preferably, on filamentous phage. The
bacteriophage expression system can be used to express Fab
fragments, Fv's with an. engineered intermolecular disulfide bond
to stabilize the V.sub.H -V.sub.L pair (dsFv's), scFvs, or diabody
fragments.
The antibody genes of the phage display libraries may be from
pre-immunized donors. For instance, the phage display library could
be a display library prepared from the spleens of mice previously
immunized with a mixture of proteins (such as a lysate of human
T-cells). Immunization can optionally be used to bias the library
to contain a greater number of recombinant antibodies reactive
towards a specific set of proteins (such as proteins found in human
T-cells). Alternatively, the library antibodies may be derived from
naive or synthetic libraries. The naive libraries have been
constructed from spleens of mice which have not been contacted by
external antigen. In a synthetic library, portions of the antibody
sequence, typically those regions corresponding to the
complementarity determining regions (CDR) loops, have been
mutagenized or randomized.
The phage display method involves batch-cloning the antibody gene
library into a phage genome as a fusion to the gene encoding one of
the phage coat proteins (pIII, pVI, or pVIII). The pill phage
protein gene is preferred. When the fusion product is expressed it
is incorporated into the mature phage coat. As a result, the
antibody is displayed as a fusion on the surface of the phage and
is available for binding and hence, selection, on a target protein.
Once a phage particle is selected as bearing an antibody-coat
protein fusion with the desired affinity towards the target
protein, the genetic material within the phage particle which
corresponds to the displayed antibody can be amplified and
sequenced or otherwise analyzed.
In a preferred embodiment, a phagemid is used as the expression
vector in the phage display procedures. A phagemid is a small
plasmid vector that carries gene III with appropriate cloning sites
and a phage packaging signal and contains both host and phage
origins of replication. The phagemid is unable to produce a
complete phage as the gene III fusion is the only phage gene
encoded on the phagemid. A viable phage can be produced by
infecting cells containing the phagemid with a helper phage
containing a defective replication origin. A hybrid phage emerges
which contains all of the helper phage proteins as well as the gene
III-rAb fusion. The emergent phage contains the phagemid DNA
only.
In a preferred embodiment of the invention, the recombinant
antibodies used in phage display methods of preparing
protein-capture agents for the arrays of the invention are
expressed as genetic fusions to the bacteriophage gene III protein
on a phagemid vector. For instance, the antibody variable regions
encoding a single-chain Fv fragment can be fused to the amino
terminus of the gene III protein on a phagemid. Alternatively, the
antibody fragment sequence could be fused to the amino terminus of
a truncated pIII sequence lacking the first two N-terminal domains.
The phagemid DNA encoding the antibody-pIII fusion is preferably
packaged into phage particles using a helper phage such as M13KO7
or VCS-M13, which supplies all structural phage proteins.
To display Fab fragments on phage, either the light or heavy (Fd)
chain is fused via its C-terminus to pIII. The partner chain is
expressed without any fusion to pIII so that both chains can
associate to form an intact Fab fragment.
Any method of selection may be used which separates those phage
particles which tu do bind the target protein from those which do
not. The selection method must also allow for the recovery of the
selected phages., Most typically, the phage particles are selected
on an immobilized target protein. Some phage selection strategies
known to those skilled in the art include the following: panning on
an immobilized antigen; panning on an immobilized antigen using
specific elution; using biotinylated antigen and then selecting on
a streptavidin resin or streptavidin-coated magnetic beads;
affinity purification; selection on Western blots (especially
useful for unknown antigens or antigens difficult to purify); in
vivo selection; and pathfinder selection. If the selected phage
particles are amplified between selection rounds, multiple
iterative rounds of selection may optionally be performed.
Elution techniques will vary depending upon the selection process
chosen, but typical elution techniques include washing with one of
the following solutions: HCl or glycine buffers; basic solutions
such as triethylamine; chaotropic agents; solutions of increased
ionic strength; or DTT when biotin is linked to the antigen by a
disulfide bridge. Other typical methods of elution include
enzymatically cleaving a protease site engineered between the
antibody and gene III, or by competing for binding with excess
antigen or excess antibodies to the antigen.
A method for producing an array of antibody fragments therefore
comprises first ill selecting recombinant bacteriophage which
express antibody fragments from a phage display library. The
recombinant bacteriophage are selected by affinity binding to a
protein which is an expression product, or fragment thereof, of a
cell or population of cells in an organism. (Iterative rounds of
selection are possible, but optional.) Next, at least one purified
sample of an antibody fragment from a bacteriophage which was
selected in the first step is produced. This antibody production
step typically entails infecting E. Coli cells with the selected
bacteriophage. In the absence of helper phage, the selected
bacteriophage then replicate as expressive plasmids without
producing phage progeny. Alternatively, the antibody fragment gene
of the selected recombinant bacteriopliage is isolated, amplified,
and then expressed in a suitable expression system. In either case,
following amplification, the expressed antibody fragment of the
selected and amplified recombinant bacteriophage is isolated and
purified. In a third step of the method, the earlier steps of phage
display selection and purified antibody fragment production are
repeated using affinity binding to different proteins which are
expression products, or fragments thereof, of the same cell or
population of cells as before until the desired plurality of
purified samples of different antibodies with different binding
pairs are produced. In a final step of the method, the antibody
fragment of each different purified sample is immobilized onto an
organic thinfilm on a separate patch on the surface of a substrate
to form a plurality of patches of antibody fragments on discrete,
known regions of the substrate surface covered by organic
thinfilm.
For instance, to generate an antibody array with antibody fragments
against known protein targets, open reading frames of the known
protein targets identified in DNA ,databases are amplified by
polymerase chain reaction and transcribed and translated in vitro
to produce proteins on which a recombinant bacteriophage expressing
single-chain antibody fragments are selected. Once selected, the
antibody fragment sequence of the selected bacteriophage is
amplified (typically using the polymerase chain method) and
recloned into a desirable expression system. The expressed antibody
fragments are purified and then printed onto organic thinfilms on
substrates to form the high density arrays.
In another embodiment of the invention, a method for producing an
array of protein-capture agents is provided which comprises first
selecting protein-capture agents from a library of protein-capture
agents, where the protein-capture agents are selected by their
affinity binding to the proteins from a cellular extract or body
fluid. Preferably, the proteins are from a cellular extract. The
proteins from the cellular extract or body fluid would typically be
immobilized prior to the selection step. Suitable methods of
immobilization such as crosslinking of the proteins to a resin are
well known to one of ordinary skill in the art. The next step of
this method comprises producing a plurality of purified samples of
the selected protein-capture agents. The protein-capture agent of
each different purified sample is immobilized onto an organic
thinfilm on a separate patch on the surface of a substrate to form
a plurality of patches of protein-capture agents on discrete, known
regions of the substrate surface covered by organic thinfilm.
This method of array preparation optionally also comprises the
additional step of biasing the library of protein-capture agents by
eliminating from the library those protein-capture agents which
bind certain proteins, such as the proteins of a second cellular
extract, wherein the protein-capture agents which are eliminated
are removed from the library by their binding affinity to those
certain proteins. This step of biasing the library may optionally
occur after the selection step by affinity binding to the protein,
but more typically, it occurs prior to that selection step. The
order of the selecting and biasing steps will depend on the nature
of the selection and elution procedures used in the method. One of
ordinary skill in the art will readily be able to determine an
appropriate series of steps.
In one embodiment of the optional step of biasing the library of
protein-capture agents, the library is biased to eliminate
protein-capture agents that recognize common proteins or proteins
of non-interest. This is typically achieved by passing the library
over an affinity surface, such as a chromatography column,
containing cross-linked proteins of non-interest. The "flowthrough"
containing protein-capture agents that did not react with the
affinity surface is collected. This procedure enriches the library
for protein-capture agents which bind proteins of interest or
proteins specific to the cell to be assayed. For instance, if the
library is derived from a specific cell type such a a human T-cell,
the library may optionally be biased by passing it over an affinity
surface which contains proteins prepared from a lysate of human
fibroblasts or bacterial proteins to enrich the library for
protein-capture agents which bind proteins specifically present in
fibroblasts.
In a preferred embodiment of the method of preparing the array of
protein-capture agents described above, the protein-capture agents
are antibody fragments displayed on do the surface of recombinant
bacteriophages and the library of protein-capture agents is a if
phage display library. Therefore, a method for producing an
antibody array comprises first selecting recombinant bacteriophage
expressing antibody fragments from a phage display library, where
the bacteriophage are selected by affinity binding to immobilized
proteins of a body fluid, or more preferably, a cellular extract.
The next step of this method comprises producing a plurality of
purified samples of antibody fragments expressed by the selected
recombinant bacteriophage. Preferably, antibody fragments which
specifically bind more than 1000 of the proteins of the cellular
extract are produced in this manner. In a final step of the method,
the antibody fragment of each different purified sample is
immobilized onto anorganic thinfilm on a separate patch on the
surface of a substrate to form a plurality of patches of antibody
fragments on discrete known regions of the substrate surface. One
specific example of this method is outlined in Example 6, below.
Again, this method optionally also comprises the additional step of
biasing the phage display library by eliminating from the library
those bacteriophage displaying antibody fragments which bind
certain proteins, such as the proteins of a second cellular
extract. The bacteriophage which are eliminated are removed from
the library by the binding affinity of their displayed antibody
fragments to the certain proteins.
For instance, a method of preparing an antibody array optionally
begins with a phage display library prepared from RNA isolated from
the spleens of mice previously immunized with a lysate of human
T-cells. The phage library is then passed over a column or affinity
surface comprising proteins from the lysates of background cells
such as human fibroblasts which have been cross-linked to a surface
or resin. The phage remaining in the flowthrough solution from the
first column/affinity surface is then passed over a second affinity
surface, such as a chromatography column, containing cross-linked
proteins prepared from a lysate of human T-cells. The flowthrough
solution from the second column/affinity surface is then discarded
since this solution contains phage which displays recombinant
antibodies that did not react with the second affinity surface.
Phage which specifically react with the second affinity surface and
remain bound to the second affinity surface are then collected by
elution. Elution can be achieved by lowered pH (2.0), increased
ionic strength, or proteolytic release by a specific proteolytic
cut site genetically engineered between the displayed recombinant
antibody and the gene III protein of the phage. In a next step of
the method, the eluted phage are separated into isolated plaques by
plating and then propagated as separate cultures. Periplasmic
fractions from the separate cultures are prepared and the
corresponding recombinant antibodies purified. The purified
recombinant antibodies are then dispensed into separate patches on
a 2-D array where they are immobilized onto an organic
thinfilm.
Methods of preparing an array of protein-capture agents where the
protein-capture agents have been selected against the proteins of a
cellular extract, or a body fluid, create arrays of protein-capture
agents where all of the binding partners of the arrays are not
initially known. The primary information provided by binding of
proteins to these types of arrays is contained in the pattern of
protein abundance. Once interesting patches on an array have been
identified by comparison of the protein expression pattern to that
of a control (for instance, it may be observed that there is a
significant increase in the amount of protein bound to a patch of
the array following exposure of a cell to a certain set of
conditions), the identity of the protein ligand binding to a
particular patch on the array can be assessed by affinity
purification of the protein ligand followed by microsequencing
and/or mass spectrometry or the like.
An alternative method for producing an array of protein-capture
agents comprises: selecting protein-capture agents from a library
of protein-capture agents, wherein the protein-capture agents are
selected by their binding affinity to proteins expressed by a cDNA
expression library; producing a plurality of purified samples of
the selected protein-capture agents; and immobilizing each
different purified protein-capture agent onto an organic thinfilm
on a separate patch on the surface of a substrate to form a
plurality of patches on discrete, known regions of the substrate
surface covered by organic thinfilm.
This method also optionally comprises the additional step of
biasing the protein-capture agent library by eliminating from the
library those protein-capture agents which bind certain proteins,
such as the proteins of a cellular extract, wherein the
protein-capture agents which are eliminated are removed from the
library by their binding affinity to said certain proteins. In most
cases, the proteins which are used to subtract protein-capture
agents from the library of protein-capture agents would be
immobilized. This step of biasing the library may optionally occur
after the selection stepby affinity binding to the proteins
expressed by the cDNA expression library, but more typically, it
occurs prior to that selection step. The order of these step will
depend on the nature of the selection and elution steps. One of
ordinary skill in the art will readily be able to determine an
appropriate series of steps. In the optional step of biasing the
library of protein-capture agents, the library is optionally biased
to eliminate protein-capture agents that recognme common proteins
or proteins of non-interest (as described above for a previous
embodiment). Preferably, the method further comprises the
additional step of identifying which individual selected
protein-capture agents bind which individual proteins expressed by
the cDNA expression library.
In another preferred embodiment of the the method, the
protein-capture agents are antibody fragments displayed on the
surface of recombinant bacteriophages and the library of
protein-capture agents is a phage display library.
For instance, one example of a method of preparing an array of
antibodies optionally begins with a phage display library prepared
from RNA isolated from the spleens of mice previously immunized
with a lysate of human T-cells. The phage library is then passed
over a column or affinity surface comprising proteins from the
lysates of background cells such as human fibroblasts which have
been cross-linked to a surface or resin. The phage remaining in the
flowthrough solution from the first column/affinity surface is then
collected. A cDNA expression library derived from message RNA
(mRNA) isolated from human T-cells is prepared in which the
expressed proteins from the expression library are genetically
fused with an expression tag (such as a six histidine tag). The
library is expanded and the tagged proteins are collectively
expressed and purified. The pool of purified, tagged proteins from
the CDNA expression library is cross-linked to an affinity surface,
such as a chromatography column. The phage display library which
passed through the first affinity surface or column is passed over
the affinity surface bearing the immobilized proteins of the cDNA
expression library. The flowthrough solution containing phage
displaying recombinant antibodies that did not react with the
affinity surface is discarded. Phage which specifically react with
the affinity surface are collected by elution achieved by lowering
the pH (2.0). Cells from the CDNA expression library are plated and
a filter lift of the colonies is made using nitrocellulose or
charged nylon filters. Reactive sites on the filter are blocked
with a standard blocking solution and the filters are probed with
the selected bacteriophage eluted off of the second column. The
phage are visualized by reaction with a monoclonal antibody
recognizing the gene VIII coat protein of the bacteriophage,
conjugated to alkaline phosphatase. Reactive sites on the filter
are cut out and the phage eluted from the filter pieces and
propagated separately. The eluted phage are separated into isolated
plaques and then propagated as separate cultures. Periplasmic
fractions from the separate cultures are prepared and the
corresponding recombinant antibodies purified. The purified
recombinant antibodies are then dispensed onto separate patches of
organic thinfilm on a 2-D array. Samples are reacted with the array
and protein ligands with interesting differential abundance
patterns (when compared to a control) are identified. Colonies on
the original plate corresponding to the phage-reactive sites on the
filter are propagated and the plasmids containing the cDNA
sequenced to identify the protein ligands reactive with the
recombinant antibodies of the phage.
In the preparation of the arrays of the invention, phage display
methods analogous to those used for antibody fragments may be used
for protein-capture agents other than antibody fragments as long as
the protein-capture agent is composed of protein and is of suitable
size to be incorporated into the phagemid or alternative vector and
expressed as a fusion with a bacteriophage coat protein. Phage
display techniques using non-antibody libraries typically make use
of some type of protein host scaffold structure which supports the
variable regions. For instance, .beta.-sheet proteins,
.alpha.-helical handle proteins, and other highly constrained
protein structures have been used as host scaffolds.
Alternative display vectors may also be used to produce the
protein-capture agents, such as antibody moieties, which are
printed on the arrays of the invention. Polysomes, stable
protein-ribosome-mRNA complexes, can be used to replace live
bacteriophage as the display vehicle for recombinant antibody
fragments or other proteins (Hanes and Pluckthun, Proc. Natl. Acad.
Sci USA, 94:4937-4942, 1997). The polysomes are formed by
preventing release of newly synthesized and correctly folded
protein from the ribosome. Selection of the polysome library is
based on binding of the antibody fragments or other proteins which
are displayed on the polysomes to the target protein. mRNA which
encodes the displayed protein or antibody having the desired
affinity for the target is then isolated. Larger libraries may be
used with polysome display than with phage display.
In still another alternative method of preparing the
protein-capture agents of the arrays of the invention, an
alternative display method of selection such as lambda display
(Mikawa et al., J. Mol. Biol., 262:21-30,1 996), bacterial display
(Georgiou et al., Nat. Biotechnol., 15:29-34, 1997) or eukaryotic
cell display may instead by used.
Furthermore, selection methods ;other than display methods may also
be used in the preparation of protein-capture agents for the arrays
of the invention. As indicated above, the protein-capture agents
may be obtained by any in vitro or in vivo selection procedure
known to those skilled in the art. In one embodiment of the
invention, protein-capture agents other than antibodies and
antibody fragments are batch selected on the protein in cellular
extracts. Such procedures generate a diversity of protein-capture
agents which are highly suitable for applications in
proteomics.
In alternative embodiments of the invention, the protein-capture
agents are partially or wholly prepared by synthetic means. If the
protein-capture agent is a protein, then methods of peptide
synthetic or protein ligation may optionally be used to construct a
protein from amino acid or polypeptide building blocks.
Protein-capture agents which are polynucleotides are readily
prepared synthetically.
(g) Uses of the Arrays
The present invention also provides methods of using the invention
arrays. In general, for a variety of applications including
proteomics and diagnostics, the methods of the invention involve
the delivery of the sample containing the proteins to be analyzed
to the arrays. After the proteins of the sample have been allowed
to interact with and become immobilized on the patches of the array
comprising protein-capture agents with the appropriate biological
specificity, the presence and/or amount of protein bound at each
patch is then determined.
Use of one of the protein-capture agent arrays of the invention may
optionally involve placing the two-dimensional array in a
flowchamber with approximately 1-10 microliters of fluid volume per
25 mm.sup.2 overall surface area. The cover over the array in the
flowchamber is preferably transparent or translucent. In one
embodiment, the cover may comprise Pyrex or quartz glass. In other
embodiments, the cover may be part of a detection system that
monitors interaction between the protein-capture agents immobilized
on the array and protein in a solution such as a cellular extract.
The flowchambers should remain filled with appropriate aqueous
solutions to preserve protein activity. Salt, temperature, and
other conditions are preferably kept similar to those of normal
physiological conditions. Proteins in a fluid solution may be
flushed into the flow chamber as desired and their interaction with
the immobilized protein-capture agents determined. Sufficient time
must be given to allow for binding between the protein-capture
agent and its binding partner to occur. The amount of time required
for this will vary depending upon the nature and tightness of the
affinity of the protein-capture agent for its binding partner. No
specialized microfluidic pumps, valves, or mixing techniques are
required for fluid delivery to the array.
Alternatively, protein-containing fluid can be delivered to each of
the patches of the array individually. For instance, in one
embodiment, the regions of the substrate surface may be
microfabricated in such a way as to allow integration of the array
with a number of fluid delivery channels oriented perpendicular to
the array surface, each one of the delivery channels terminating at
the site of an individual protein-capture agent-coated patch.
The sample which is delivered to the array will typically be a
fluid. In a preferred embodiment of the invention, the sample is a
cellular extract or a body fluid. The sample to be assayed may
optionally comprise a complex mixture of proteins, including a
multitude of proteins which are not binding partners of the
protein-capture agents of the array. If the proteins to be analyzed
in the sample are membrane proteins, then those proteins will
typically need to be solubilized prior to administration of the
sample to the array. If the proteins to be assayed in the sample
are proteins secreted by a population of cells in an organism, a
sample which is derived from a body fluid is preferred. If the
proteins to be assayed in the sample are intracellular, a sample
which is a cellular extract is preferred. In one embodiment of the
invention, the array may comprise protein-capture agents which bind
fragments of the expression products of a cell or population of
cells in an organism. In such a case, the proteins in the sample to
be assayed may have been prepared by performing a digest of the
protein in a cellular extract or a body fluid. In an alternative
application of the array, the proteins from only specific fractions
of a cell are collected for analysis in the sample.
In general, delivery of solutions containing proteins to be bound
by the protein-capture agents of the array may optionally be
preceded, followed, or accompanied by delivery of a blocking
solution. A blocking solution contains protein or another moiety
which will adhere to sites of non-specific binding on the array.
For instance, solutions of bovine serum albumin or milk may be used
as blocking solutions.
It is understood that some proteins a sample which are not the
intended binding partner of the protein-capture agents of a patch
(and may, in fact, be the intended binding partner of another
patch) on the array may still bind to the patch to some degree.
Preferably, this type of binding only occurs to a very minor
degree. Also, it is understood that even when the correct binding
partners are present in the solution being assayed, the binding
partners will bind to the patch comprising their protein-capture
agent with less than 100% efficiency.
A wide range of detection methods is applicable to the methods of
the invention. As desired, detection may be either quantitative or
qualitative. The invention array can be interfaced with optical
detection methods such as absorption in the visible or infrared
range, chemoluminescence, and fluorescence (including lifetime,
polarization, fluorescence correlation spectroscopy (FCS), and
fluorescence-resonance energy transfer (FRET)). Furthermore, other
modes of detection such as those based on optical waveguides PCT
Publication (WO 96/26432 and U.S. Pat. No. 5,677,196), surface,
plasmon resonance, surface charge sensors, and surface force
sensors are compatible with many embodiments of the invention.
Alternatively, technologies such as those based on Brewster Angle
microscopy (BAM) (Schaaf et al., Langmuir, 3:1131-1135 (1987)) and
ellipsometry (U.S. Pat. Nos. 5,141,311 and 5,116,121; Kim,
Macromolecules, 22:2682-2685 (1984)) could be applied. Quartz
crystal microbalances and desorption processes (see for example,
U.S. Pat. No. 5,719,060) provide still other alternative detection
means suitable for at least some embodiments of the invention
array. An example of an optical biosensor system compatible both
with some arrays of the present invention and a variety of
non-label detection principles including surface plasmon resonance,
total internal reflection fluorescence (TIRF), Brewster Angle
microscopy, optical waveguide lightmode spectroscopy (OWLS),
surface charge measurements, and ellipsometry can be found in U.S.
Pat. No. 5,313,264.
Although non-label detection methods are generally preferred, some
of the types of detection methods commonly used for traditional
immunoassays which require the use of labels may be applied to the
arrays of the present invention. These techniques include
noncompetitive immunoassays, competitive immunoassays, and dual
label, ratiometric immunoassays. These particular techniques are
primarily suitable for use with the arrays of protein-capture
agents when the number of different protein-capture agents with
different specificity is small (less than about 100). In the
competitive method, binding-site occupancy is determined
indirectly. In this method, the protein-capture agents of the array
are exposed to a labeled developing agent, which is typically a
labeled version of the analyte or an analyte analog. The developing
agent competes for the binding sites on the protein-capture agent
with the analyte. The fractional occupancy of the protein-capture
agents on different patches can be determined by the binding of the
developing agent to the protein-capture agents of the individual
patches. In the noncompetitive method, binding site occupancy is
determined directly. In this method, the patches of the array are
exposed to a labeled developing agent capable of binding to either
the bound analyte or the occupied binding sites on the
protein-capture agent. For instance, the developing agent may be a
labeled antibody directed against occupied sites (ie;, a "sandwich
assay"). Altematively, a dual label, ratiometric, approach may be
taken where the protein-capture agent is labeled with one label and
the second, developing agent is labeled with a second label (Ekins,
et al., Clinica Chimica Acta., 194:91-114, 1990). Many different
labeling methods may be used in the aforementioned techniques,
including radioisotopic, enzymatic, chemiluminescent, and
fluorescent methods. Fluorescent methods are preferred.
FIG. 8 shows a schematic diagram of one type of fluorescence
detection unit which may be used to monitor interaction of
immobilized protein-capture agents of an array with a protein
analyte. In the illustrated detection unit, the array of
protein-capture agents 21 is positioned on a base plate 20. Light
from a 100 W mercury arc lamp 25 is 1, directed through an
excitation filter 24 and onto a beam splitter 23. The light is then
directed through a lens 22, such as a Micro Nikkor 55 mm 1:2:8
lens, and onto the array 21. Fluorescence emission from the array
returns through the lens 22 and the beam splitter 23. After next
passing through an emission filter 26, the emission is received by
a cooled CCD camera 27, such as the Slowscan TE/CCD-1024SF&SB
(Princeton Instruments). The camera is operably connected to a CPU
28 which is in turn operably connected to a VCR 29 and a monitor
30.
FIG. 9 shows a schematic diagram of an alternative detection method
based on ellipsometry. Ellipsometry allows for information about
the sample to be determined from the observed change in the
polarization state of a reflected light wave. Interaction of a
protein analyte with a layer of immobilized protein-capture agents
on a patch results in a thickness change and alters the
polarization status of a plane-polarized light beam reflected off
the surface. This process can be monitored in situ from aqueous
phase and, if desired, in imaging mode. In a typical setup,
monochromatic light (e.g. from a He--Ne laser, 30) is plane
polarized (polarizer 31) and directed onto the surface of the
sample and detected by a detector 35. A compensator 32 changes the
elliptically polarized reflected beam to plane-polarized. The
corresponding angle is determined by an analyzer 33 and then
translated into the ellipsometric parameters Psi and Delta which
change upon binding of protein with the protein-capture agents.
Additional information can be found in Azzam, et al., Ellipsometry
and Polarized Light, North-Holland Publishing Company: a Amsterdam,
1977.
The arrays of the present invention are particularly useful for
proteomics. Those arrays which comprise significant numbers of
protein-capture agents of different specificity on separate patches
can bind significant numbers of proteins which are expression
products, or fragments thereof, of a cell or population of cells in
an organism and are particularly suitable for use in applications
involving proteomics. For instance, an array with at least about
10.sup.3 and up to about 10.sup.5 different protein-capture agents
such as antibodies or antibody fragments can provide a highly
comprehensive picture of the protein content of the cell under a
specific set of conditions.
In one embodiment of the invention, a method of assaying in
parallel for a plurality of different proteins in a sample which
are expression products, or fragments thereof, of a cell or a
population of cells in an organism, is provided which comprises the
following steps: first, delivering the sample to an array of
spatially distinct patches of different protein-capture agents
under conditions suitable for protein binding, wherein each of the
proteins being assayed is a binding partner of the protein-capture
agent of at least one patch on the array; next, optionally washing
said array to remove unbound or nonspecifically bound components of
the sample from the array; and in a final step, detecting, either
directly or indirectly, for the presence or amount of protein bound
to each patch of the array.
In another embodiment of the invention, a method of assaying in
parallel for a plurality of different proteins in a sample which
are expression products, or fragments thereof, of a cell or a
population of cells in an organism, comprises first delivering the
sample to the invention array of protein-capture agents under
conditions suitable for protein binding, wherein each of the
proteins being assayed is a binding partner of the protein-capture
agent of at least one patch on the array. The first step may be
followed by an optional step of washing the array with fluid to
remove unbound or nonspecifically bound components of the sample
from the array. Lastly, the presence or amount of protein bound to
each patch is detected, either directly or indirectly.
A variety of different embodiments of the invention array of
protein-capture agents may be used in the methods for assaying in
parallel for a plurality of different proteins in a sample which
are expression products, or fragments thereof, of a cell or a
population of cells in an organism. Generally, preferred
embodiments of these methods comprise the use of preferred arrays
of the invention. For instance, in preferred embodiments of the
method, the protein-capture agents are antibodies or antibody
fragments. In firer preferred embodiments for assaying the
different amounts of a plurality of proteins in a cell in parallel
or the protein expression pattern of a cell, the plurality of
patches on the array can bind at least about 100 or at least about
103 different proteins which are the expression products, or
fragments thereof, of a cell or population of cells in an organism.
Alternatively, the plurality of patches on the array used in the
methods can bind at least about 10.sup.4 different proteins which
are the expression products, or fragments thereof, of a cell or
population of cells in an organism.
The methods of assaying in parallel for a plurality of different
proteins in a sample which are expression products, or fragments
thereof, of a cell or a population of cells in an organism,
optionally comprise the additional step of further characterizing
the protein bound to at least one patch of the array. This step is
typically designed to identify the nature of the protein bound to
the protein-capture agent of a particular patch. In some cases, the
entire identity of the bound protein may not be known and the
purpose of the further characterization may be the initial
identification of the mass, sequence, structure and/or activity of
the bound protein. In other cases, the basic identity of the
protein may be known, but the post-translational modification,
activation state, or some other feature of the protein may not be
known. In one embodiment, the step of further characterizing the
proteins involves measuring the activity of the proteins. Although
in some cases it may be preferable to remove the protein from the
patch before the step of further characterizing the protein is
carried out, in other cases the protein can be further
characterized while still bound to the patch. In still further
embodiments, the protein-capture agents of the patch which binds a
protein can be used to isolate and/or purify the protein from
cells. The purified sample can then be characterized through
traditional means such as microsequencing, mass spectrometry, and
the like.
In another embodiment, the present invention provides a method of
determining the protein expression pattern of a cell or population
of cells in an organism. This method involves first delivering a
sample containing expression products, or fragments thereof, of the
cell or population of cells to the protein-capture agent array of
the invention under conditions suitable for protein binding. The
presence and/or amount of protein bound to each patch can then be
determined by a suitable detection means. The detection may be
either direct or indirect. Quantitative detection is typically
preferred for this application (and for other proteomics
applications). The method preferably further comprises an
additional step before the detection step comprising washing the
array to remove unbound or nonspecifically bound components of the
sample from the array. The amount of protein bound to a patch of
the array may optionally be determined relative to the amount of a
second protein bound to a second patch of the array. The method of
determining the protein expression pattern of a cell or a
population of cells in an organism, optionally comprises the
additional step of further characterizing the proteins bound to at
least one patch of the array, as previously described above.
In the method of assaying the protein expression pattern of a cell
or population of cells in an organism, many of the targets of the
protein-capture agents of the array may optionally be of unknown
sequence, identity, and/or function. For instance, the antibodies
of the array may have been prepared by selecting a phage display
library by affinity binding to the immobilized proteins of a
cellular extract which contains many unidentified proteins. If the
protein bound by a protein-capture agent on a particular patch of
an array is unknown, but is of interest, then that protein may
optionally be later identified or characterized by first using the
same protein-capture agent that was used on the array to isolate
the protein in question from cells. The isolated binding partner
from the cell can then be assayed directly for function and/or
sequenced.
The arrays of protein-capture agents may also be used to compare
the protein expression patterns of two cells or populations of
cells. In this method, a sample containing expression products, or
fragments thereof, of a first cell or population of cells is
delivered to the invention array of protein-capture agents under
conditions suitable for protein binding. In an analogous manner, a
sample containing expression products, or fragments therof, of a
second cell or population of cells to a second array, is delivered
to a second array which is identical to the first array.
Preferably, both arrays are then washed to remove unbound or
nonspecifically bound components of the sample from the arrays. In
a final step, the amounts of protein remaining bound to the patches
of the first array are compared to the amounts of protein remaining
bound to the corresponding patches of the second array. If it is
desired to determine the differential protein expression pattern of
two cells or populations of cells, for instance, then the amount of
protein bound to the patches of the first array may be subtracted
from the amount of protein bound to the corresponding patches of
the second array.
Methods of comparing the protein expression of two cells or
populations of cells are particularly useful for the understanding
of biological processes. For instance, using these methods, the
protein expression patterns of identical cells or closely related
cells exposed to different conditions can be compared. Most
typically, the protein content of one cell or population of cells
is compared to the protein content of a control cell or population
of cells. For instance, in one embodiment of the invention, one of
the cells or populations of cells is neoplastic and the other cell
is not. In another embodiment, one of the two cells or populations
of cells being assayed is infected with a pathogen. Alternatively,
one of the two cells or populations of cells has been exposed to a
stressor and the other cell or population of cells serves as a
control. The stressor may optionally be chemical, environmental, or
thermal. One of the two cells may optionally be exposed to a drug
or a potential drug and its protein expression pattern compared to
a control cell.
Such methods of assaying differential gene expression at the
protein level are useful in the identification and validation of
new potential drug targets as well as for drug screening. For
instance, the method may be used to identify a protein which is
overexpressed in tumor cells, but not in normal cells. This protein
may be a target for drug intervention. Inhibitors to the action of
the overexpressed protein can then be developed. Alternatively,
antisense strategies to inhibit the overexpression may be
developed. In another instance, the protein expression pattern of a
cell, or population of cells, which has been exposed to a drug or
potential drug can be compared to that of a cell, or population of
cells, which has not been exposed to the drug. This comparison will
provide insight as to whether or not the drug has had the desired
effect on a target protein (drug efficacy) and whether other
proteins of the cell, or population of cells, have also been
affected (drug specificity).
The arrays of the present invention are also suitable for
diagnostic applications and suitable for use in diagnostic devices.
The high density of the antibodies on some arrays of the present
invention enables a large number of different, antibody-based
diagnostic tests to be formatted onto a single biochip. The
protein-capture agents on the invention array can be used to
evaluate the status of a disease condition in a tissue, such as a
tumor, where the expression levels of certain proteins in the cells
of the tissue is known to be indicative of a particular type of
disease condition or stage of a disease condition. If certain
patterns of protein expression are not previously known to be
indicative of a disease state, the protein-capture agent arrays of
the invention can then first be used to establish this
information.
Accordingly, in one embodiment, the invention provides a method of
evaluating a disease condition in a tissue of an organism
comprising first contacting the invention array of protein-capture
agents with a sample comprising the expression products, or
fragments thereof, of the cells of the tissue being evaluated,
wherein the contacting occurs under conditions suitable for protein
binding and wherein the binding partners of a plurality of
protein-capture agents on the array include proteins which are
expression products, or fragment thereof of the cells of the tissue
and whose expression levels are indicative of the disease
condition. The method next comprises detecting, either directly or
indirectly, for the presence of protein to each patch. In a
preferred embodiment, the method further comprises the step of
washing the array to remove unbound or nonspecifically bound
components of the sample from the array. In such a method, the
array will typically comprise protein-capture agents which bind
those proteins whose presence, absence, or relative amount in cells
is known to be indicative of a particular type of disease condition
or state of a disease condition. For instance, the plurality of
proteins being assayed in such a method may include such proteins
as HER2 protein or prostate-specific antigen (PSA).
(h) EXAMPLES
The following specific examples are intended to illustrate the
invention and should not be construed as limiting the scope of the
claims:
Example 1
Fabrication of a Two-dimensional Array by Photolithography.
In a preferred embodiment of the invention, two-dimensional arrays
are fabricated onto the substrate material via standard
photolithography and/or thin film deposition. Alterative techniques
include microcontact printing. Usually, a computer-aided design
pattern is transferred to a photomask using standard techniques,
which is then used to transfer the pattern onto a silicon wafer
coated with photoresist.
In a typical example, the array ("chip") with lateral dimensions of
10.times.10 mm comprises squared patches of a bioreactive layer
(here: gold as the coating on a silicon substrate) each
0.1.times.0.1 mm in size and separated by hydrophobic surface areas
with a 0.2 mm spacing. 4" diameter Si(100) wafers (Virginia
Semiconductor) are used as bulk materials. Si(100) wafers are first
cleaned in a 3:1 mixture of H.sub.2 SO.sub.4, conc.: 30% H.sub.2
O.sub.2 (90.degree. C., 10 min), rinsed with deionized water (18
M.OMEGA.cm), finally passivated in 1% aqueous HF, and singed at
150.degree. C. for 30 min to become hydrophobic. The wafer is then
spincoated with photoresist (Shipley 1813), prebaked for 25 minutes
at 90.degree. C., exposed using a Karl Suss contact printer and
developed according to standard protocols. The wafer is then dried
and postbaked at 110.degree. C. for 25 min. In the next step, the
wafer is primed with a titanium layer of 20 nm thickness, followed
by a 200 nm thick gold layer. Both layers were deposited using
electron-beam evaporation (5 .ANG./s). After resist stripping and a
short plasma treatment, the gold patches can be further chemically
modified to achieve the desired bioreactive and biocompatible
properties (see Example 3, below).
Example 2
Fabrication of a Two-dimensional Array by Deposition Through a Hole
Mask.
In another preferred embodiment the array of gold patches is
fabricated by thin film deposition through a hole mask which is in
direct contact with the substrate. In a typical example, Si(100)
wafers are first cleaned in a 3:1 mixture of H.sub.2 SO.sub.4,
conc.: 30% H.sub.2 O.sub.2 (90.degree. C., 10 min), rinsed with
deionized water (18 M.OMEGA.cm), finally passivated in 1%. aqueous
HF and singed at 150.degree. C. for 30 min to become hydrophobic.
The wafer is then brought into contact with a hole mask exhibiting
the positive pattern of the desired patch array. In the next step,
the wafer is primed with a titanium layer of 20 nm thickness,
followed by a 200 nm thick gold layer. Both layers were deposited
using electron-beam evaporation (5 .ANG./s). After removal of the
mask, the gold patches can be further chemically modified to
achieve the desired bioreactive and biocompatible properties (see
Example 3, below).
Example 3
Synthesis of an Aminoreactive Monolayer Molecule (Following the
Procedure Outlined in Wagner el al., Biophys. J., 1996,
70:2052-2066).
General. .sup.1 H- and .sup.13 C-NMR spectra are recorded on Bruker
instruments (100 to 400 MHz). Chemical shifts (.delta.) are
reported in ppm relative to internal standard ((CH.sub.3).sub.4 Si,
.delta.=0.00 (.sup.1 H- and .sup.13 C-NMR)). FAB-mass spectra are
recorded on a VG-SABSEQ instrument (Cs.sup.+, 20 keV). Transmission
infrared spectra are obtained as dispersions in KBr on an FTIR
Perkin-Elmer 1600 Series instrument. Thin-layer chromatography
(TLC) is performed on precoated silica gel 60 F254 plates (MERCK,
Darmstadt, FRG), and detection was done using Cl.sub.2 /toluidine,
PdCl.sub.2 and UV-detection under NH.sub.3 -vapor. Medium pressure
liquid chromatography (MTLC) is performed on a Labomatic MD-80
(LABOMATIC INSTR. AG, Allschwil, Switzerland) using a Buechi column
(460.times.36 mm; BUECHI, Flawil, Switzerland), filled with silica
gel 60 (particle size 15-40 .mu.m) from Merck.
Synthesis of 11,11'-dithiobis(succinimidylundecanoate) (DSU).
Sodium thiosulfate (55.3 g, 350 mmol) is added to a suspension of
11-bromo-undecanoic acid (92.8 g, 350 mmol) in 50% aqueous
1,4-dioxane (1000 ml). The mixture is heated at reflux (90.degree.
C.) for 2 h until the reaction to the intermediate Bunte salt was
complete (clear solution). The oxidation to the corresponding
disulfide is carried out in situ by adding iodine in portions until
the solution retained with a yellow to brown colour. The surplus of
iodine is retitrated with 15% sodium pyrosulfite in water. After
removal of 1,4-dioxane by rotary evaporation the creamy suspension
is filtered to yield product 11,11'-dithiobis(undecanoicacid).
Recrystallization from ethyl acetate/THF provides a white solid
(73.4 g, 96.5%): mp 94.degree. C.; .sup.1 H NMR (400 MHz,
CDCl.sub.3 /CD.sub.3 OD 95:5): .delta. 2.69 (t, 2H, J=7.3 Hz), 2.29
(t, 2H, J=7.5 Hz), 1.76-1.57 (m, 4H), and 1.40-1.29 (m, 12H);
FAB-MS (Cs.sup.+, 20 keV): nt/z (relative intensity) 434 (100,
M.sup.+). Anal. Calcd. for C.sub.22 H.sub.42 O.sub.4 S.sub.2 : C,
60.79; H, 9.74; S, 14.75. Found: C, 60.95; H, 9.82; S, 14.74. To a
solution of 11,11'-dithiobis(undecanoic acid) (1.0 g, 2.3 mmol) in
THF (50 ml) is added N-hydroxysuccinimide (0.575 g, 5 minol)
followed by DCC (1.03 g, 5 mmol) at 0.degree. C. After the reaction
mixture is allowed to warm to 23.degree. C. and is stirred for 36 h
at room temperature, the dicyclohexylurea (DCU) is filtered.
Removal of the solvent under reduced pressure and recrystallization
from acetone/hexane provides
11,11'-dithiobis(succinimidylundecanoate) as a white solid. Final
purification is achieved by medium pressure liquid chromatography
(9 bar) using silica gel and a 2:1 mixture of ethyl acetate and
hexane. The organic phase is concentrated and dried in vacuum to
afford 11,11'-dithiobis(succinimidylundecanoate) (1.12 g, 78%): mp
95.degree. C.; .sup.1 H NMR (400 MHz, CDCl.sub.3): .delta. 2.83 (s,
4H), 2.68 (t, 2H, J=7.3 Hz), 2.60 (t, 2H, J=7.5 Hz), 1.78-1.63 (m,
4H), and 1.43-1.29 (m, 12H); FAB-MS (Cs.sup.+, 20 keV): m/z
(relative intensity) 514 (100), 628 (86, M.sup.+). Anal. Calcd. for
C.sub.30 H.sub.48 N.sub.2 O.sub.8 S.sub.2 : C, 57.30; H, 7.69; N,
4.45; S, 10.20. Found: C, 57.32; H, 7.60; N, 4.39; S, 10.25.
Example 4
Formation of an Aminoreactive Monolayer on Gold (Following the
Procedure of Wagner et al., Biophys. J., 1996, 70:2052-2066).
Monolayers based on 11,11'-dithiobis(succinimidylundecanoate) (DSU)
can be deposited on Au(111) surfaces of substrates described under
Examples 1 and 2 by immersing them into a 1 mM solution of DSU in
chloroform at room temperature for 1 hour. After rinsing with 10
volumes of solvent, the N-hydroxysuccinimidyl-terminated monolayer
is dried under a stream of nitrogen and immediately used for
immobilization of the protein-capture agents.
Example 5
Formation and Use of an Array of Immobilized Fab' Antibody
Fragments to Detect Concentrations of Soluble Proteins Prepared
from Cultured Mammalian Cells.
Collections of IgG antibodies are purchased from commercial sources
(e.g. Pierce, Rockford, Ill.). The antibodies are first purified by
affinity chromatography based on binding to immobilized protein A.
The antibodies are diluted 1:1 in binding buffer(0.1 M Tris-HCl,
0.15 M NaCl, pH 7.5). A 2 ml minicoluin containing a gel with
immobilized protein A is prepared. (Hermanson, et. al., Immobilized
Affinity Ligand Techniques, Academic Press, San Diego, 1992.) The
column is equilibrated with 10 ml of binding buffer. Less than 10
mg of immunoglobulin is applied to each 2 ml minicolumn and the
column is washed with binding buffer until the absorbance at 280 nm
is less than 0.02. The bound immunoglobulins are eluted with 0.1 M
glycine, 0.15 M NaCl, pH 2.8, and immediately neutralized with 1.0
M Tris-HCl, pH 8.0 to 50 mM final concentration and then dialyzed
against 10 mM sodium phosphate, 0.15 M NaCl, pH 7.2 and stored at
4.degree. C.
The purified immunoglobulin are digested with immobilized pepsin.
Pepsin is an acidic endopeptidase and hydrolyzes proteins favorably
adjacent to aromatic and dicarboxylic L-amino acid residues.
Digestion of IgG with pepsin generates intact F(ab').sub.2
fragments. Immobilized pepsin gel is washed with digestion buffer;
20 mM sodium acetate, pH 4.5. A solution of purified IgG at 10
mg/ml is added to the immobilized pepsin gel and incubated at
37.degree. C. for 2 hours. The reaction is neutralized by the
addition of 10 mM Tris-HCl, pH 7.5 and centrifuged to pellet the
gel. The supernatants liquid is collected and applied to an
immobilized protein A column, as described above, to separate the
F(ab').sub.2 fragments from the Fc and undigested IgG. The pooled
F(ab').sub.2 is dialyzed against 10 mM sodium phosphate, 0.15 M
NaCl, pH 7.2 and stored at 4.degree. C. The quantity of pooled,
eluted F(ab').sub.2 is measured by peak area absorbance at 280
nm.
The purified F(ab').sub.2 fragments at a concentration of 10 mg/ml
are reduced at 37.degree. C. for 1 hour in a buffer of 10 mM sodium
phosphate, 0.15 M NaCl, 10 mM 2-mercaptoethylamine, 5 mM EDTA, pH
6.0. The Fab' fragments are separated from unsplit F(ab').sub.2
fragments and concentrated by application to a Sephadex G-25 column
(M.sub.r =46,000-58,000). The pooled Fab' fragments are dialyzed
against 10 mM sodium phosphate, 0.15 M NaCl pH 7.2. The reduced
Fab' fragments are diluted to 100 .mu.g/ml and applied onto the
bioreactive patches containing exposed aminoreactive functional
groups using a computer-aided, capillary-based microdispensing
system (for antibody immobilization procedures, see Dammer et al.,
Biophys. J., 70:2437-2441, 1996). After an immobilization period of
30 minutes at 30.degree. C., the array is rinsed extensively with
10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.
Transformed human cells grown in culture are collected by low speed
centrifugation, briefly washed with ice-cold phosphate-buffered
solution (PBS), and then resuspended in ice-cold hypotonic buffer
containing DNase/RNase (10 .mu.g/ml each, final concentration) and
a mixture of protease inhibitors. Cells are transferred to a
microcentrifuge tube, allowed to swell for 5 minutes, and lysed by
rapid freezing in liquid nitrogen and thawing in ice-cold water.
Cell debris and precipitates are removed by high-speed
centrifugation and the supernatants is cleared by passage through a
0.45 .mu.m filter. The cleared lysate is applied to the Fab'
fragment array described above and allowed to incubate for 2 hours
at 30.degree. C. After binding the array is washed extensively with
10 mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0. The
location and amount of bound proteins are determined by optical
detection.
Example 6
Formation and Use of an Array of Immobilized Antibody Fragments to
Detect Concentrations of Soluble Proteins Prepared from Cultured
Mammalian Cells.
A combinatorial library of filamentous phage expressing scFv
antibody fragments is generated based on the technique of
McCafferty and coworkers; McCafferty, et al., Nature, 1990,
348:552-554; Winter and Milstein, Nature, 1991, 349:293-299.
Briefly, mRNA is purified from mouse spleens and used to construct
a cDNA library. PCR fragments encoding sequences of the variable
heavy and light chain immunoglobulin genes of the mouse are
amplified from the prepared cDNA. The amplified PCR products are
joined by a linker region of DNA encoding the 15 amino acid peptide
(Gly.sub.4 SerGly.sub.2 CysGlySerGly.sub.4 Ser) (SEQ ID NO: 1) and
the resulting full-length PCR fragment is cloned into an expression
plasmid (pCANTAB 5 E) in which the purification peptide tag (E Tag)
has been replaced by a His.sub.6 peptide (SEQ ID NO: 2).
Electrocompetent TG1 E.coli cells are transformed with the
expression plasmid by electroporation. The pCANTAB-transformed
cells are induced to produced functional in filamentous phage
expressing scFv fragments by superinfection with M13KO7 helper
phage. Cells are grown on glucose-deficient medium containing the
antibiotics ampicillin (to select for cells with the phagemid) and
kanamycin (to select for cells infected with M13KO7). In the
absence of glucose, the lac promoter present on the phagemid is no
longer repressed, and synthesis of the scFv-gene 3 fusion
begins.
Proteins from a cell lysate are adsorbed to the wells of a 96-well
plate. Transformed human cells grown in culture are collected by
low speed centrifugation and the cells are briefly washed with
ice-cold PBS. The washed cells are then resuspended in ice-cold
hypotonic buffer containing DNaselRNase (10 .mu.g/ml each, final
concentration) and a mixture of protease inhibitors, allowed to
swell for 5 minutes, and lysed by rapid freezing in liquid nitrogen
and thawing in ice-cold water. Cell debris and precipitates are
removed by high-speed centrifugation and the supernatant is cleared
by passage through a 0.45 .mu.m filter. The cleared lysate is
diluted to 10 .mu.g/ml in dilution buffer; 20 mM PIPES, 0.15 M
NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT,
pH 7.2 and applied to the 96-plate wells. After immobilization for
1 hour at 30.degree. C., the well is washed with the dilution
buffer and then incubated with dilution buffer containing 10%
nonfat dry milk to block unreacted sites. After the blocking step,
the well is washed extensively with the dilution buffer.
Phage expressing displayed antibodies are separated from E. coli
cells by centrifugation and then precipitated from the supernatant
by the addition of 15% w/v PEG 8000, 2.5 M NaCl followed by
centrifugation. The purified phage are resuspended in the dilution
buffer containing 3% nonfat dry milk and applied to the well
containing the immobilized proteins described above, and allowed to
bind for 2 hours at 37.degree. C., followed by extensive washing
with the binding buffer. Phage are eluted from the well with an
elution buffer; 20 mM PIPES, 1 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA,
5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. The well is then
extensively washed with purge buffer; 20 mM PIPES, 2.5 M NaCl, 0.1%
CHAPS, 10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2.
The well is then extensively washed with dilution buffer; 20 mM
PIPES, 0.15 M NaCl, 0.1% CHAPS, 10%, 5 mM EDTA, 5 mM
2-mercaptoethanol, 2 mM DTT, pH 7.2. The eluted phage solution is
then re-applied to a new well containing adsorbed antigen and the
panning enrichment is repeated 4 times. Finally, the phage are
eluted from the well with 2M of NaCl in 20 mM PIPES, 0.1% CHAPS,
10%, 5 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM DTT, pH 7.2. Eluates
are collected and mixed with log-phase TG1 cells, and grown at
37.degree. C. for 1 hour and then plated onto SOB medium containing
ampicillin and glucose and allowed to grow for 12-24 hours.
Individual colonies are picked and arrayed into 96-well 2 ml blocks
containing SOB medium and M13K07 helper phage and grown for 8 hours
with shaking at 37.degree. C. The phage are separated from cells by
centrifugation and precipitated with PEG/NaCl as described above.
Concentrated phage are used to infect HB2151 E. coli. E. coli TG1
produces a suppressor tRNA which allows readthrough (suppression)
of an amber stop codon located between the scFv and phage gene 3
sequences of the pCANTAB 5 E plasmid. Infected HB2151 cells are
selected on medium containing ampicillin, glucose, and nalidixic
acid. Cells are grown to mid-log and then centrifuged and
resuspended in medium lacking glucose and growth continued. Soluble
scFv fragments will accumulate in the cell periplasm. A periplasmic
extract is prepared from pelleted cells by mild osmotic shock. The
soluble scFv released into the supernatant is purified by affinity
binding to Ni-NTA activated agarose and eluted with 10 mM EDTA
The purified scFv antibody fragments are diluted to 100 .mu.g/ml
and applied onto the bioreactive patches with exposed aminoreactive
groups using a computer-aided, capillary-based microdispensing
system. After an immobilization period of 30 minutes at 30.degree.
C., the array is rinsed extensively with 10 mM sodium phosphate,
0.15 M NaCl, 5 mM EDTA, pH 7.0.
Transformed human cells grown in culture are collected by low speed
centrifugation, briefly washed with ice-cold PBS, and then
resuspended in ice-cold hypotonic buffer containing DNase/RNase (10
.mu.g/ml each, final concentration) and mixture of protease
inhibitors. Cells are transferred to a microcentrifuige tube,
allowed to swell for 5 minutes, and lysed by rapid freezing in
liquid nitrogen and thawing in ice-cold water. Cell debris and
precipitates are removed by high-speed centrifugation and the
supernatant is cleared by passage through a 0.45 .mu.m filter. The
cleared lysate is applied to the scFv fragment array described
above and allowed to incubate for 2 hours at 30.degree. C. After
binding, the array is washed extensively with 0.1 M sodium
phosphate, 0.15 M NaCl, 5 mM EDTA pH 7.0. The location and amount
of bound proteins are determined by optical detection.
Patterns of binding are established empirically by testing
dilutions of a control cell extract. Extracts from experimental
cells are diluted to a series of concentrations and then tested
against the array. Patterns of protein expression in the
experimental cell lysates are compared to protein expression
patterns in the control samples to identify proteins with unique
expression profiles.
Example 7
Formation and Use of an Array of Immobilized Monoclonal Antibodies
to Detect Concentrations of Soluble Proteins Prepared from Cultured
Mammalian Cells.
Collections of monoclonal antibodies are purchased from commercial
suppliers as either raw ascities fluid or purified by
chromatography over protein A, protein G, or protein L. If from raw
ascites fluid, the antibodies are purified using a HiTrap Protein G
or HiTrap Protein A column (Pharmacia) as appropriate for the
immunoglobulin subclass and species. Prior to chromatography the
ascites are diluted with an equal volume of 10 mM sodium phosphate,
0.9% NaCl, pH 7.4 (PBS) and clarified by passage through a 0.22
.mu.m filter. The filtrate is loaded onto the column in PBS and the
column is washed with two column volumes of PBS. The antibody is
eluted with 100 mM Glycine-HCl, pH 2.7 (for protein G) or 100 mM
citric acid, pH 3.0 (for protein A). The eluate is collected into
1/10 volume 1 M Tris-HCl, pH 8.0. The final pH is 7.5. Fractions
containing the antibodies are confirmed by SDS-PAGE and then pooled
and dialyzed against PBS.
The different samples of purified antibodies are each diluted to
100 .mu.g/ml. Each different antibody sample is applied to a
separate patch of an array of aminoreactive monolayer patches (see
Example 4, above) using a computer-aided, capillary-based
microdispensing system. After an immobilization period of 30
minutes at 30.degree. C., the array is rinsed extensively with 10
mM sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 7.0.
Transformed human cells grown in culture are collected by low speed
centrifugation, briefly washed with ice-cold PBS, and resuspended
in ice-cold hypotonic buffer containing Dnase/Rnase (10 .mu.g/ml
each, final concentration) and a mixture of protease inhibitors.
Cells are transferred to a microcentrifuge tube, allowed to swell
for 5 minutes, and lysed by rapid freezing in liquid nitrogen and
thawing in ice-cold water. Cell debris and precipitates are removed
by high-speed centrifugation and the supernatant is cleared by
passage through a 0.45 .mu.m filter. The cleared lysate is applied
to the monoclonal antibody array described above and allowed to
incubate for 2 hours at 30.degree. C. After binding the array is
washed extensively as in Example 6, above. The location and amount
of bound proteins are determined by optical detection.
All documents cited in the above specification are herein
incorporated by reference. In addition, the copending U.S. patent
application "Arrays of Proteins and Methods of Use Thereof", filed
on Jul. 14, 1999, with the identifier 24406-0004 P1, for the
inventors Peter Wagner, Dana Ault-Riche, Steffen Nock, and
Christian Itin, is herein incorporated by reference in its
entirety. Various modifications and variations of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in the art are
intended to be within the scope of the following claims.
* * * * *
References